This document reports on a study of the oxygenation properties of hemoglobin from the earthworm Lumbricus terrestris under varying conditions of pH, salts, and temperature. Key findings include:
1) Hemoglobin from L. terrestris exhibits relatively small cooperativity (free energy of 1.6-2.8 kcal/mol) and a large, pH-dependent Hill coefficient that reaches a maximum of 7.9.
2) Cations, not anions, control oxygen binding, with divalent cations having a larger effect than monovalent cations. Effectiveness decreases in the order Ca2+ > Sr2+ > Ba2+ and Mg2+ > Na+.
Nitrate Reductase Complex of Escherichia coli K-12: Participation of Specific...IPN
1) The study examined the components involved in nitrate reduction by Escherichia coli, including formate dehydrogenase and cytochrome b1.
2) Certain E. coli mutants unable to reduce nitrate had low or undetectable levels of formate dehydrogenase activity, suggesting distinct formate dehydrogenases are involved in nitrate reduction and hydrogen formation.
3) Five mutants formed gas when grown without nitrate and had benzyl viologen-linked formate dehydrogenase, while four mutants formed little gas and lacked multiple formate dehydrogenase activities, further supporting distinct formate dehydrogenases.
1. The document describes a new catalyst (1) that promotes the kinetic resolution of racemic alcohols through enantioselective acylation at ambient temperature. Using just 5 mol% of the catalyst results in recovery of optically active alcohols with 92-99% ee.
2. Investigation of the reaction mechanism suggests catalyst 1 acts through an "induced fit" mechanism like natural enzymes. NMR studies show the catalyst exists in an "open conformation" that is catalytically active, but transforms to a "closed conformation" in the reactive intermediate that controls stereoselectivity.
3. The design of catalyst 1 places stereocontrolling chiral centers far from the active site to avoid comprom
This document describes a new method for site-specifically labeling proteins using genetically encoded norbornene and tetrazine probes. Specifically:
- A norbornene-containing amino acid was genetically encoded in E. coli and mammalian cells using the pyrrolysyl tRNA synthetase system.
- A series of tetrazine probes were developed that react rapidly and specifically with norbornenes via a Diels-Alder reaction.
- The labeling of encoded norbornene was shown to be specific and much faster than other bioorthogonal reactions, demonstrating advantages for protein labeling in vitro and on cells.
- Rapid and site-specific labeling of a cell surface protein was demonstrated,
OBC epoxidations paper - Queen Mary University LONDON UK - Thomas FollierThomas Follier
This document reports on a study of the catalytic activity of manganese complexes with two similar polyamine ligands (7 and 8) that differ by the presence of a secondary or tertiary nitrogen, in the epoxidation of styrene. Ligand 7 showed the highest activity with MnSO4 and H2O2, while ligand 8 was most effective with Mn(OTf)2, MnCl2, and Mn(ClO4)2 using peracetic acid. Kinetic analysis indicated the structural differences in the ligands lead to differences in the nature of the active species formed. Ligand 7 with MnSO4 produced the epoxide in 78% yield, while ligand 8 with Mn(OT
Sipma, 2004, Effect Of Carbon Monoxide, Hydrogen And Sulfate On Thermophilic ...roelmeulepas
This document summarizes a study on the conversion of carbon monoxide (CO) by two anaerobic sludge samples at 55°C. The study aimed to elucidate the conversion routes and determine the effect of substrate (CO) concentration and the presence of hydrogen gas. Inhibition experiments showed CO conversion occurred via a hydrogenogenic population producing hydrogen and carbon dioxide, with the products then used by methanogens, acetogens, or sulfate reducers depending on sludge source and inhibitors. Both sludges could produce hydrogen from CO, indicating potential for biological hydrogen production from synthesis gas containing CO. The paper mill sludge was also capable of sulfate reduction using hydrogen produced from high CO concentrations, showing CO-rich synthesis gas can efficiently
This document summarizes a study examining the role of serum albumin in catalyzing the formation of S-nitrosothiols (RS-NO) from nitric oxide (NO) in vitro and in vivo. The key findings are:
1) At physiological NO concentrations, plasma albumin becomes saturated with NO and accelerates the formation of low-molecular-weight RS-NO through micellar catalysis of NO oxidation within its hydrophobic core and specific transfer of NO+ to low-molecular-weight thiols.
2) Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating low-molecular-weight thiols.
3
The document summarizes characterization of the H134C mutant of the Thermus thermophilus Rieske protein. Key findings include:
1) The H134C mutant substitutes one ligating histidine for a cysteine, changing the iron-sulfur cluster ligation from 2Cys-2His to 3Cys-1His.
2) Studies using UV-visible spectroscopy, circular dichroism, and reaction with diethyl pyrocarbonate (DEPC) show the H134C mutant is stable across a wide pH range, unlike the similar mitochondrial protein mitoNEET.
3) Reaction of H134C with DEPC results in modification of the cluster but no reduction,
The document discusses testing the reactivity between various Good's buffers and vernadite (δ-Mn(IV)O2). X-ray diffraction analysis found structural modifications to vernadite when reacted with EPPS, MOPSO, HEPES, and MOPS buffers, indicating partial reduction of Mn(IV) to Mn(III). The inertness of BES may be due to its molecular geometry lacking nitrogen-containing rings found in the other buffers. Diffraction patterns of vernadite reacted with varying concentrations of MOPS and EPPS showed stacking order was not directly proportional to buffer concentration.
Nitrate Reductase Complex of Escherichia coli K-12: Participation of Specific...IPN
1) The study examined the components involved in nitrate reduction by Escherichia coli, including formate dehydrogenase and cytochrome b1.
2) Certain E. coli mutants unable to reduce nitrate had low or undetectable levels of formate dehydrogenase activity, suggesting distinct formate dehydrogenases are involved in nitrate reduction and hydrogen formation.
3) Five mutants formed gas when grown without nitrate and had benzyl viologen-linked formate dehydrogenase, while four mutants formed little gas and lacked multiple formate dehydrogenase activities, further supporting distinct formate dehydrogenases.
1. The document describes a new catalyst (1) that promotes the kinetic resolution of racemic alcohols through enantioselective acylation at ambient temperature. Using just 5 mol% of the catalyst results in recovery of optically active alcohols with 92-99% ee.
2. Investigation of the reaction mechanism suggests catalyst 1 acts through an "induced fit" mechanism like natural enzymes. NMR studies show the catalyst exists in an "open conformation" that is catalytically active, but transforms to a "closed conformation" in the reactive intermediate that controls stereoselectivity.
3. The design of catalyst 1 places stereocontrolling chiral centers far from the active site to avoid comprom
This document describes a new method for site-specifically labeling proteins using genetically encoded norbornene and tetrazine probes. Specifically:
- A norbornene-containing amino acid was genetically encoded in E. coli and mammalian cells using the pyrrolysyl tRNA synthetase system.
- A series of tetrazine probes were developed that react rapidly and specifically with norbornenes via a Diels-Alder reaction.
- The labeling of encoded norbornene was shown to be specific and much faster than other bioorthogonal reactions, demonstrating advantages for protein labeling in vitro and on cells.
- Rapid and site-specific labeling of a cell surface protein was demonstrated,
OBC epoxidations paper - Queen Mary University LONDON UK - Thomas FollierThomas Follier
This document reports on a study of the catalytic activity of manganese complexes with two similar polyamine ligands (7 and 8) that differ by the presence of a secondary or tertiary nitrogen, in the epoxidation of styrene. Ligand 7 showed the highest activity with MnSO4 and H2O2, while ligand 8 was most effective with Mn(OTf)2, MnCl2, and Mn(ClO4)2 using peracetic acid. Kinetic analysis indicated the structural differences in the ligands lead to differences in the nature of the active species formed. Ligand 7 with MnSO4 produced the epoxide in 78% yield, while ligand 8 with Mn(OT
Sipma, 2004, Effect Of Carbon Monoxide, Hydrogen And Sulfate On Thermophilic ...roelmeulepas
This document summarizes a study on the conversion of carbon monoxide (CO) by two anaerobic sludge samples at 55°C. The study aimed to elucidate the conversion routes and determine the effect of substrate (CO) concentration and the presence of hydrogen gas. Inhibition experiments showed CO conversion occurred via a hydrogenogenic population producing hydrogen and carbon dioxide, with the products then used by methanogens, acetogens, or sulfate reducers depending on sludge source and inhibitors. Both sludges could produce hydrogen from CO, indicating potential for biological hydrogen production from synthesis gas containing CO. The paper mill sludge was also capable of sulfate reduction using hydrogen produced from high CO concentrations, showing CO-rich synthesis gas can efficiently
This document summarizes a study examining the role of serum albumin in catalyzing the formation of S-nitrosothiols (RS-NO) from nitric oxide (NO) in vitro and in vivo. The key findings are:
1) At physiological NO concentrations, plasma albumin becomes saturated with NO and accelerates the formation of low-molecular-weight RS-NO through micellar catalysis of NO oxidation within its hydrophobic core and specific transfer of NO+ to low-molecular-weight thiols.
2) Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating low-molecular-weight thiols.
3
The document summarizes characterization of the H134C mutant of the Thermus thermophilus Rieske protein. Key findings include:
1) The H134C mutant substitutes one ligating histidine for a cysteine, changing the iron-sulfur cluster ligation from 2Cys-2His to 3Cys-1His.
2) Studies using UV-visible spectroscopy, circular dichroism, and reaction with diethyl pyrocarbonate (DEPC) show the H134C mutant is stable across a wide pH range, unlike the similar mitochondrial protein mitoNEET.
3) Reaction of H134C with DEPC results in modification of the cluster but no reduction,
The document discusses testing the reactivity between various Good's buffers and vernadite (δ-Mn(IV)O2). X-ray diffraction analysis found structural modifications to vernadite when reacted with EPPS, MOPSO, HEPES, and MOPS buffers, indicating partial reduction of Mn(IV) to Mn(III). The inertness of BES may be due to its molecular geometry lacking nitrogen-containing rings found in the other buffers. Diffraction patterns of vernadite reacted with varying concentrations of MOPS and EPPS showed stacking order was not directly proportional to buffer concentration.
The [2,3]-Wittig rearrangement is a special class of [2,3]-sigma tropic rearrangement which involves an α-oxy carbanions as the migrating terminus to afford various types of homoallylic alcohols.
This document reports on research measuring the strength of silicon-hydrogen bonds in various silane compounds using photoacoustic calorimetry. The main findings are:
1) Silicon-hydrogen bond strengths are significantly weakened by the successive substitution of silyl groups, with the bond in tris(trimethylsilyl)silane having one of the weakest bond strengths measured at 79.0 kcal/mol.
2) Steric effects and radical stabilization may contribute to the weakening of the silicon-hydrogen bond from additional silyl substitutions, though the exact origin is unclear.
3) The weak silicon-hydrogen bond in tris(trimethylsilyl)silane
This doctoral thesis uses computational methods like density functional theory and molecular dynamics simulations to study the structural and functional role of cytochrome P450 enzymes. It investigates the metabolism of various substrates by CYP3A4 and CYP450 enzymes to understand reaction pathways and influence of substrate structure on reactivity. Specific reactions studied include hydroxylation of phenyl rings, morpholine rings, and camphor. Flexibility studies using the RIGIX program also examined how the protein environment modulates electronic structure and reactivity. The research provides new insights into CYP450 catalysis at the molecular level and could aid in drug design.
This document describes a study on using degraded polyethylene terephthalate (PET) waste to remove metal ions from aqueous solutions. PET waste was degraded using glycolysis to produce low molecular weight degradation products. The degradation product was characterized and identified as bis(2-hydroxyethyl) terephthalate (BHET). Batch experiments showed that BHET had the highest affinity for removing lead and cadmium ions from solutions. The removal of metal ions was found to depend on factors like pH, initial metal ion concentration, and the size and charge of the metal ion species. FTIR spectroscopy and DSC analysis provided evidence that metal ions interacted with functional groups on the BHET through coordinate bonding.
Computational and Experimental Studies of MTO Catalyzed Olefin HydrogenationKaram Idrees
The poster that I presented at the 253rd American Chemical Society National Meeting and Exposition in San Francisco,
CA. It highlights some of my REU research at North Carolina State University under the mentorship of Dr. Elon Ison.
Ion water interaction biophysical journalmganguly123
The document discusses how the charge density of ions affects their strength of hydration and interactions in biological structures. It finds that small, highly charged ions (kosmotropes) strongly bind water molecules, while large monovalent ions of low charge density (chaotropes) weakly bind water. Crystalline salts dissolve exothermically only when one ion is a kosmotrope and the other is a chaotrope. This suggests kosmotropes and chaotropes preferentially form ion pairs in solution. The major intracellular ions—phosphate and carboxylate anions and potassium/arginine cations—behave as kosmotropes and chaotropes, respectively, allowing them
This document summarizes a study on the synthesis and NMR analysis of various Naproxen and Ibuprofen ester derivatives. Several diastereoisomeric Naproxyl-Naproxenate, Naproxyl-Ibuprofenate, and Ibuprofyl-Naproxenate esters were synthesized. Variable temperature NMR experiments showed differences in aromatic ring dynamics between esters, likely due to varying axial rotation speeds. Chiral shift reagent NMR experiments were also performed and provided insights into epimerization levels, assignments of proton signals, and geometries of the ester complexes.
The document discusses the Petersen matrix notation for describing complex biochemical systems with multiple simultaneous reactions. It provides an example of the Petersen matrix for the Activated Sludge Model 1 (ASM1), which models the key biological processes in activated sludge wastewater treatment. The ASM1 Petersen matrix includes 13 components, 8 processes, and kinetic equations for each process. It allows calculating the rate of change for each component based on the effects of all processes. The document also provides details on the components, processes, stoichiometry, and kinetic equations included in the ASM1.
This document summarizes experiments investigating the roles of various organic additives in facilitating coupling reactions between haloarenes and arenes using potassium tert-butoxide (KOtBu). Amino acids like sarcosine and proline were found to initiate these reactions via electron transfer, forming aryl radicals. Secondary amino acids were more effective than primary or tertiary amino acids. Piperazinedione derivatives formed from the condensation of amino acids were also found to initiate reactions by forming electron-rich enolates or dianions that act as electron donors. Alcohols, 1,2-diols, and 1,2-diamines were also investigated for their ability to form electron donors that initiate these coupling reactions.
tuning the pH Response of i-Motif DNA Oligonucleotides_Lannes_et_al-2015-Chem...saheli halder
This document discusses tuning the pH response of i-motif DNA oligonucleotides. The authors introduced 5-methylcytosines (5-MeC) and 5-bromocytosines (5-BrC) into the human telomeric i-motif sequence to shift its pH response range. They found that 5-MeC shifted the pH response towards more basic values, while 5-BrC shifted it towards more acidic values. Additionally, lengthening the sequence shifted the pH response in a more basic direction. The modifications did not thermally destabilize the i-motifs. 5-BrC substitution led to a ten-fold increase in folding kinetics compared to the other sequences.
The document summarizes the setup of a metabolic model to analyze cellular reactions in E. coli during mixed acid fermentation. It defines substrates, products, biomass constituents, and intracellular metabolites. It then lists the specific stoichiometric equations for glucose conversion and outlines the metabolic model matrix with glucose as the substrate and various products and intracellular metabolites. The model is analyzed to obtain information on ATP synthesis rates and redox balance from consumption of NADH based on experimental yields of metabolic products per glucose.
Molecular dynamics-of-ions-in-two-forms-of-an-electroactive-polymerDarren Martin Leith
This document summarizes molecular dynamics simulations of two forms of an electroactive polymer interacting with ions. In one simulation, an amphiphilic polymer forms a charged monolayer interface between a vacuum and an aqueous layer containing ions. The stability of the monolayer under hydrostatic pressure and charge imbalance is investigated. In another simulation, a polythiophene oligomer is twisted into a helix serving as an ion channel between two aqueous regions separated by a phospholipid bilayer membrane.
Organocatalysts for enantioselective synthesis of fine chemicals: definitions, trends and developments
This document provides a summary of organocatalysis and its use in the enantioselective synthesis of fine chemicals. Organocatalysis refers to the use of small organic molecules to catalyze organic transformations. There are two main types of organocatalysis - covalent organocatalysis where the catalyst forms a covalent bond with the substrate, and non-covalent organocatalysis involving hydrogen bonding or ion pair formation. Examples of successful organocatalytic reactions discussed include the proline-catalyzed aldol reaction and the synthesis of oseltamivir using enamine catalysis. The field
This document summarizes research exploring the biochemical properties and remediation applications of an unusual P450 system (XplA/B) found in Rhodococcus bacteria that is able to degrade the explosive compound RDX. Key findings include:
1) XplA has a high affinity for RDX (Kd = 58 μM) and XplB functions as its native reductase partner to efficiently degrade RDX.
2) Expression of both XplA and XplB in Arabidopsis plants enables significantly faster removal of RDX from contaminated liquid culture and soil compared to plants expressing only XplA.
3) Under anaerobic conditions, RDX degradation by XplA/B produces
This document summarizes the efficient synthesis of novel 3-aminocyclobut-2-en-1-ones, which are potent antagonists of VLA-4. The synthesis involves the condensation of cyclobuta-1,3-diones with a phenylalanine-derived primary amine. This produces vinylogous amide derivatives in good yields. The cyclobuta-1,3-diones are prepared through cycloaddition of ketene intermediates, generated from acid chlorides, with various alkynes. The resulting 3-aminocyclobut-2-en-1-ones can then be functionalized at C-2 through reaction with electrophilic reagents
Enzymes catalyze biochemical reactions and are characterized by their catalytic activity. The document discusses several key aspects of enzyme mechanisms and kinetics, including:
1) Enzymes have an active site that binds substrates and facilitates chemical reactions through proximity, orientation, and catalytic groups like metal ions or amino acid residues.
2) Michaelis-Menten kinetics describe the relationship between substrate concentration, reaction rate, and parameters like Vmax and Km.
3) Common metalloenzymes contain catalytic metal ions like zinc, copper, iron and manganese that participate in redox reactions.
This document provides details about Organic Chemistry II, a 2 unit course taught over two hours per week. The course content includes stereochemistry, functional group chemistry, substitution and elimination reactions. The document also provides a detailed lecture note on substitution reactions of alkanes, including halogenation reactions and their mechanisms via a free radical chain mechanism. It discusses the relative reactivities and stabilities of primary, secondary and tertiary positions during halogenation. Finally, it provides an overview of stereochemistry, including a discussion of enantiomers and chirality around tetrahedral carbons.
We here introduced and elucidated the mechanism of hydrolysis reaction of Adenosine Triphosphate (ATP) molecules for generation of bio-energy in the living Systems, in which the detained reaction processes of hydrolysis of ATP molecules and its properties, The center effects and functions of ATP molecules in the life activity, AIP enzyme and production of bio-energy in the hydrolysis of ATP molecules and The formation of ATP molecules and its relation with Δ-μH+ were described and explained. From these introductions and results we understand and knew that this mechanism of generation of bio-energy is its an important form, which exists widely in the life bodies of generation of bio-energy. Therefore, to elucidate and research this mechanism and its properties have quite important meanings in biology, biochemistry and biophysics.
This summary provides the key points from the document in 3 sentences:
The document discusses evidence that some meteorites, including the Murchison meteorite, contain amino acids that preferentially exhibit left-handed chirality, suggesting life's preference for left-handed amino acids may have an extraterrestrial origin. While this finding is intriguing, it is difficult to definitively prove the excess is indigenous to the meteorites and not due to terrestrial contamination. New research on the Murchison meteorite helps address this issue by examining nitrogen isotope ratios of individual amino acid enantiomers, finding left-handed excesses that are too high to include much terrestrial material.
This document summarizes the development of a mild and efficient method for synthesizing symmetrical and unsymmetrical azo compounds from aromatic anilines using copper bromide (CuBr) and N-methylmorpholine N-oxide (NMO) as an oxidizing system. Optimization experiments showed that CuBr with 1 equivalent of NMO in an acetonitrile/water solvent mixture gave the best yields. A variety of substituted anilines underwent homocoupling to produce symmetrical azo compounds in good to excellent yields. Unsymmetrical azo compounds were also synthesized via cross-coupling of different anilines in moderate to good yields. The reaction was shown to proceed via single electron transfer from Cu(I) to NMO
Circular dichroism is the difference in absorption of left and right circularly polarized light by a chiral molecule. It occurs due to interactions between the molecule's chiral chromophores and polarized light. CD spectroscopy is used to analyze the secondary structure of proteins and monitor structural changes. The technique provides structural signatures for alpha helices, beta sheets, and random coils. It is a powerful tool for studying protein folding and structural changes under various conditions.
Circular dichroism spectroscopy measures the differential absorption of left and right circularly polarized light by chiral molecules. When light passes through an optically active substance, the left and right circular polarizations are absorbed to different extents. A CD spectrometer contains a light source, monochromator, polarizer, photoelastic modulator and detector. It measures the CD signal as a function of wavelength, providing information about secondary structure of proteins and nucleic acids. CD spectroscopy requires minimal sample amounts and can quickly analyze secondary structure without crystallization. It is useful for studying protein folding, ligand binding and environmental effects on structure.
The [2,3]-Wittig rearrangement is a special class of [2,3]-sigma tropic rearrangement which involves an α-oxy carbanions as the migrating terminus to afford various types of homoallylic alcohols.
This document reports on research measuring the strength of silicon-hydrogen bonds in various silane compounds using photoacoustic calorimetry. The main findings are:
1) Silicon-hydrogen bond strengths are significantly weakened by the successive substitution of silyl groups, with the bond in tris(trimethylsilyl)silane having one of the weakest bond strengths measured at 79.0 kcal/mol.
2) Steric effects and radical stabilization may contribute to the weakening of the silicon-hydrogen bond from additional silyl substitutions, though the exact origin is unclear.
3) The weak silicon-hydrogen bond in tris(trimethylsilyl)silane
This doctoral thesis uses computational methods like density functional theory and molecular dynamics simulations to study the structural and functional role of cytochrome P450 enzymes. It investigates the metabolism of various substrates by CYP3A4 and CYP450 enzymes to understand reaction pathways and influence of substrate structure on reactivity. Specific reactions studied include hydroxylation of phenyl rings, morpholine rings, and camphor. Flexibility studies using the RIGIX program also examined how the protein environment modulates electronic structure and reactivity. The research provides new insights into CYP450 catalysis at the molecular level and could aid in drug design.
This document describes a study on using degraded polyethylene terephthalate (PET) waste to remove metal ions from aqueous solutions. PET waste was degraded using glycolysis to produce low molecular weight degradation products. The degradation product was characterized and identified as bis(2-hydroxyethyl) terephthalate (BHET). Batch experiments showed that BHET had the highest affinity for removing lead and cadmium ions from solutions. The removal of metal ions was found to depend on factors like pH, initial metal ion concentration, and the size and charge of the metal ion species. FTIR spectroscopy and DSC analysis provided evidence that metal ions interacted with functional groups on the BHET through coordinate bonding.
Computational and Experimental Studies of MTO Catalyzed Olefin HydrogenationKaram Idrees
The poster that I presented at the 253rd American Chemical Society National Meeting and Exposition in San Francisco,
CA. It highlights some of my REU research at North Carolina State University under the mentorship of Dr. Elon Ison.
Ion water interaction biophysical journalmganguly123
The document discusses how the charge density of ions affects their strength of hydration and interactions in biological structures. It finds that small, highly charged ions (kosmotropes) strongly bind water molecules, while large monovalent ions of low charge density (chaotropes) weakly bind water. Crystalline salts dissolve exothermically only when one ion is a kosmotrope and the other is a chaotrope. This suggests kosmotropes and chaotropes preferentially form ion pairs in solution. The major intracellular ions—phosphate and carboxylate anions and potassium/arginine cations—behave as kosmotropes and chaotropes, respectively, allowing them
This document summarizes a study on the synthesis and NMR analysis of various Naproxen and Ibuprofen ester derivatives. Several diastereoisomeric Naproxyl-Naproxenate, Naproxyl-Ibuprofenate, and Ibuprofyl-Naproxenate esters were synthesized. Variable temperature NMR experiments showed differences in aromatic ring dynamics between esters, likely due to varying axial rotation speeds. Chiral shift reagent NMR experiments were also performed and provided insights into epimerization levels, assignments of proton signals, and geometries of the ester complexes.
The document discusses the Petersen matrix notation for describing complex biochemical systems with multiple simultaneous reactions. It provides an example of the Petersen matrix for the Activated Sludge Model 1 (ASM1), which models the key biological processes in activated sludge wastewater treatment. The ASM1 Petersen matrix includes 13 components, 8 processes, and kinetic equations for each process. It allows calculating the rate of change for each component based on the effects of all processes. The document also provides details on the components, processes, stoichiometry, and kinetic equations included in the ASM1.
This document summarizes experiments investigating the roles of various organic additives in facilitating coupling reactions between haloarenes and arenes using potassium tert-butoxide (KOtBu). Amino acids like sarcosine and proline were found to initiate these reactions via electron transfer, forming aryl radicals. Secondary amino acids were more effective than primary or tertiary amino acids. Piperazinedione derivatives formed from the condensation of amino acids were also found to initiate reactions by forming electron-rich enolates or dianions that act as electron donors. Alcohols, 1,2-diols, and 1,2-diamines were also investigated for their ability to form electron donors that initiate these coupling reactions.
tuning the pH Response of i-Motif DNA Oligonucleotides_Lannes_et_al-2015-Chem...saheli halder
This document discusses tuning the pH response of i-motif DNA oligonucleotides. The authors introduced 5-methylcytosines (5-MeC) and 5-bromocytosines (5-BrC) into the human telomeric i-motif sequence to shift its pH response range. They found that 5-MeC shifted the pH response towards more basic values, while 5-BrC shifted it towards more acidic values. Additionally, lengthening the sequence shifted the pH response in a more basic direction. The modifications did not thermally destabilize the i-motifs. 5-BrC substitution led to a ten-fold increase in folding kinetics compared to the other sequences.
The document summarizes the setup of a metabolic model to analyze cellular reactions in E. coli during mixed acid fermentation. It defines substrates, products, biomass constituents, and intracellular metabolites. It then lists the specific stoichiometric equations for glucose conversion and outlines the metabolic model matrix with glucose as the substrate and various products and intracellular metabolites. The model is analyzed to obtain information on ATP synthesis rates and redox balance from consumption of NADH based on experimental yields of metabolic products per glucose.
Molecular dynamics-of-ions-in-two-forms-of-an-electroactive-polymerDarren Martin Leith
This document summarizes molecular dynamics simulations of two forms of an electroactive polymer interacting with ions. In one simulation, an amphiphilic polymer forms a charged monolayer interface between a vacuum and an aqueous layer containing ions. The stability of the monolayer under hydrostatic pressure and charge imbalance is investigated. In another simulation, a polythiophene oligomer is twisted into a helix serving as an ion channel between two aqueous regions separated by a phospholipid bilayer membrane.
Organocatalysts for enantioselective synthesis of fine chemicals: definitions, trends and developments
This document provides a summary of organocatalysis and its use in the enantioselective synthesis of fine chemicals. Organocatalysis refers to the use of small organic molecules to catalyze organic transformations. There are two main types of organocatalysis - covalent organocatalysis where the catalyst forms a covalent bond with the substrate, and non-covalent organocatalysis involving hydrogen bonding or ion pair formation. Examples of successful organocatalytic reactions discussed include the proline-catalyzed aldol reaction and the synthesis of oseltamivir using enamine catalysis. The field
This document summarizes research exploring the biochemical properties and remediation applications of an unusual P450 system (XplA/B) found in Rhodococcus bacteria that is able to degrade the explosive compound RDX. Key findings include:
1) XplA has a high affinity for RDX (Kd = 58 μM) and XplB functions as its native reductase partner to efficiently degrade RDX.
2) Expression of both XplA and XplB in Arabidopsis plants enables significantly faster removal of RDX from contaminated liquid culture and soil compared to plants expressing only XplA.
3) Under anaerobic conditions, RDX degradation by XplA/B produces
This document summarizes the efficient synthesis of novel 3-aminocyclobut-2-en-1-ones, which are potent antagonists of VLA-4. The synthesis involves the condensation of cyclobuta-1,3-diones with a phenylalanine-derived primary amine. This produces vinylogous amide derivatives in good yields. The cyclobuta-1,3-diones are prepared through cycloaddition of ketene intermediates, generated from acid chlorides, with various alkynes. The resulting 3-aminocyclobut-2-en-1-ones can then be functionalized at C-2 through reaction with electrophilic reagents
Enzymes catalyze biochemical reactions and are characterized by their catalytic activity. The document discusses several key aspects of enzyme mechanisms and kinetics, including:
1) Enzymes have an active site that binds substrates and facilitates chemical reactions through proximity, orientation, and catalytic groups like metal ions or amino acid residues.
2) Michaelis-Menten kinetics describe the relationship between substrate concentration, reaction rate, and parameters like Vmax and Km.
3) Common metalloenzymes contain catalytic metal ions like zinc, copper, iron and manganese that participate in redox reactions.
This document provides details about Organic Chemistry II, a 2 unit course taught over two hours per week. The course content includes stereochemistry, functional group chemistry, substitution and elimination reactions. The document also provides a detailed lecture note on substitution reactions of alkanes, including halogenation reactions and their mechanisms via a free radical chain mechanism. It discusses the relative reactivities and stabilities of primary, secondary and tertiary positions during halogenation. Finally, it provides an overview of stereochemistry, including a discussion of enantiomers and chirality around tetrahedral carbons.
We here introduced and elucidated the mechanism of hydrolysis reaction of Adenosine Triphosphate (ATP) molecules for generation of bio-energy in the living Systems, in which the detained reaction processes of hydrolysis of ATP molecules and its properties, The center effects and functions of ATP molecules in the life activity, AIP enzyme and production of bio-energy in the hydrolysis of ATP molecules and The formation of ATP molecules and its relation with Δ-μH+ were described and explained. From these introductions and results we understand and knew that this mechanism of generation of bio-energy is its an important form, which exists widely in the life bodies of generation of bio-energy. Therefore, to elucidate and research this mechanism and its properties have quite important meanings in biology, biochemistry and biophysics.
This summary provides the key points from the document in 3 sentences:
The document discusses evidence that some meteorites, including the Murchison meteorite, contain amino acids that preferentially exhibit left-handed chirality, suggesting life's preference for left-handed amino acids may have an extraterrestrial origin. While this finding is intriguing, it is difficult to definitively prove the excess is indigenous to the meteorites and not due to terrestrial contamination. New research on the Murchison meteorite helps address this issue by examining nitrogen isotope ratios of individual amino acid enantiomers, finding left-handed excesses that are too high to include much terrestrial material.
This document summarizes the development of a mild and efficient method for synthesizing symmetrical and unsymmetrical azo compounds from aromatic anilines using copper bromide (CuBr) and N-methylmorpholine N-oxide (NMO) as an oxidizing system. Optimization experiments showed that CuBr with 1 equivalent of NMO in an acetonitrile/water solvent mixture gave the best yields. A variety of substituted anilines underwent homocoupling to produce symmetrical azo compounds in good to excellent yields. Unsymmetrical azo compounds were also synthesized via cross-coupling of different anilines in moderate to good yields. The reaction was shown to proceed via single electron transfer from Cu(I) to NMO
Circular dichroism is the difference in absorption of left and right circularly polarized light by a chiral molecule. It occurs due to interactions between the molecule's chiral chromophores and polarized light. CD spectroscopy is used to analyze the secondary structure of proteins and monitor structural changes. The technique provides structural signatures for alpha helices, beta sheets, and random coils. It is a powerful tool for studying protein folding and structural changes under various conditions.
Circular dichroism spectroscopy measures the differential absorption of left and right circularly polarized light by chiral molecules. When light passes through an optically active substance, the left and right circular polarizations are absorbed to different extents. A CD spectrometer contains a light source, monochromator, polarizer, photoelastic modulator and detector. It measures the CD signal as a function of wavelength, providing information about secondary structure of proteins and nucleic acids. CD spectroscopy requires minimal sample amounts and can quickly analyze secondary structure without crystallization. It is useful for studying protein folding, ligand binding and environmental effects on structure.
Circular dichroism (CD) spectroscopy measures the difference in absorbance of left and right circularly polarized light by a substance. CD occurs when asymmetrical molecules interact with polarized light. A CD spectrometer measures the CD spectra of proteins and nucleic acids to determine their secondary structure composition and monitor conformational changes. Key applications include estimating protein and nucleic acid structure, studying macromolecular interactions, and characterizing folding and unfolding kinetics and thermodynamics.
This document discusses optical rotatory dispersion (ORD), which is defined as the rate of change of specific rotation with a change in wavelength. ORD is used to determine the structures of carbonyl compounds. Key points covered include: the principles of plane polarized light, optical activity, specific rotation, and circular birefringence; factors that affect specific rotation; Cotton effects and curves; and the octant rule for establishing absolute configuration from ORD data.
Optical rotatory dispersion is the variation in the optical rotation of a substance with a change in the wavelength of light.
For wavelengths that are absorbed by the optically active sample, the two circularly polarized components will be absorbed to differing extents. This unequal absorption is known as circular dichroism.
This document provides an overview of optical rotatory dispersion (ORD) and circular dichroism (CD). It defines ORD as the rate of change of specific rotation with changing wavelength, and explains that CD occurs when chiral compounds absorb left and right circularly polarized light to different extents. The document outlines several types of ORD and CD curves and describes applications like qualitative analysis, enzyme evaluation, and studying the structure of proteins and nucleic acids. It compares the key differences between ORD and CD techniques and briefly describes the instrumentation used for each.
The document discusses optical rotatory dispersion (ORD) and circular dichroism. It begins by introducing Sujit R. Patel from the Department of Pharmaceutics and provides an overview of topics to be covered including ORD, the ORD curve, circular dichroism, and the octant rule. It then discusses the fundamentals of ORD, how specific rotation changes with wavelength, and the different types of ORD curves including plain and anomalous curves. It also addresses the cotton effect seen in anomalous dispersion curves. Finally, it covers applications of circular dichroism spectroscopy such as determining protein and nucleic acid conformation.
This document summarizes the rational design and generation of a catalytic antibody that selectively hydrolyzes a specific substrate. Researchers designed an antibody to bind a transition state analogue for the hydrolysis of a carbonate substrate. They generated monoclonal antibodies against a nitrophenyl phosphonate transition state analogue. One antibody was found to catalyze the hydrolysis of the carbonate substrate, displaying Michaelis-Menten kinetics. The antibody-catalyzed reaction had substrate specificity and was competitively inhibited by the corresponding phosphate transition state analogue, demonstrating the ability to rationally design catalytic antibodies.
The document summarizes the results of a study that measured the standard enthalpies of ionization of 21 substituted anilinium ions using calorimetry. It found that both the enthalpies and entropies of ionization correlated well with the Gibbs free energies of ionization. The experimental results were analyzed according to Hepler's theory of substituent effects and compared to predictions of electrostatic theory.
1) Boronic acids were shown to be potent competitive inhibitors of the nitrile hydratase enzyme from Pseudonocardia thermophila (PtNHase).
2) X-ray crystallography revealed that the sulfenic acid ligand of the active site cysteine attacks and forms a covalent bond with the boron atom of bound boronic acid inhibitors, representing a potential reaction intermediate.
3) A new mechanism is proposed for nitrile hydration by nitrile hydratases wherein the sulfenic acid ligand acts as the catalytic nucleophile.
This document summarizes a study that measured the enthalpy change (ΔH) associated with the α-helix to random coil transition of an alanine peptide in water using calorimetry. The researchers synthesized a 50-residue peptide containing primarily alanine residues and determined its ΔH to be between 0.9-1.3 kcal/mol per residue, providing a basic parameter for predicting thermal unfolding of peptide helices. Circular dichroism spectra and melting curves confirmed the peptide adopted an α-helical structure at low temperatures and underwent a reversible helix-coil transition. The ΔH value suggests the peptide backbone, rather than side chains, makes the dominant contribution to helix stability.
This document describes the synthesis and characterization of a calcium phosphonate framework material (Ca-PiPhtA) and its derivatives for proton conductivity applications. The parent framework Ca-PiPhtA-I was synthesized from calcium chloride and the ligand 5-(dihydroxyphosphoryl)isophthalic acid under acidic aqueous conditions, producing a structure with 1D channels and high water content. Upon heating or exposure to ammonia vapors, it undergoes partial dehydration or structural transformations to form new derivatives Ca-PiPhtA-II and Ca-PiPhtA-NH3 respectively, as characterized through methods such as X-ray diffraction and thermal analysis. Proton conductivity measurements found the materials conduct
The students cultivated two cultures of sulfur-utilizing bacteria from pond water using different reactor designs. They recorded pH, alkalinity, and optical density over six days as indicators of sulfur oxidation activity. Visual observation showed growth of pink/orange bacteria in the photosynthetic reactor and grey/orange bacteria in the non-photosynthetic reactor, pointing to growth of sulfur-utilizing Chromatiales and Thiobacillus bacteria. The data trends suggested the sulfur substrate was depleted after two days, explaining the stability of measured values over time.
This document summarizes research on the binding mechanism of Cu(II) ions to a poly-histidine peptide found in the venom of an African viper. The peptide contains 9 histidine residues and effectively binds copper, forming thermodynamically stable complexes. Experimental techniques like potentiometric titrations, UV-Vis, and EPR spectroscopy as well as molecular dynamics simulations were used to study the copper binding sites and structure of the complexes. The results show that copper prefers binding to histidine residues separated by one amino acid in the peptide and that more stable complexes form when the peptide adopts an alpha-helical structure.
1) A new fluorescent receptor (compound 3) was designed and synthesized based on an acridine-imidazolium functionalized cholestane for anion sensing.
2) Binding studies using UV-vis and fluorescence spectroscopy showed that compound 3 has the highest selectivity for hydrogen pyrophosphate (HP2O7^3-) with a binding constant of 1.5 x 10^4 M^-1.
3) Molecular modeling showed that HP2O7^3- binds more strongly to compound 3 than other anions through hydrogen bonding interactions between its oxygen atoms and amino and imidazolium hydrogen atoms on the receptor.
The document describes a proposed study to develop a novel class of photoswitchable carboxylic acids whose acidity can be reversibly controlled by light. Dithienylethene compounds will be synthesized that can switch between open and closed states with different light wavelengths. The closed state is expected to be more conjugated, allowing electron donating/withdrawing substituents to influence acidity. Quantum calculations and acidity comparisons will predict acidity changes between states. Target compounds will be synthesized and characterized, with acidity measurements used to validate light-controlled changes. If successful, these photoswitchable acids could enable new applications in catalysis, biochemistry, and more.
This document summarizes the key stages and discoveries in photosynthesis research:
1. Moll's half leaf experiment in the 1700s showed that CO2 is required for photosynthesis.
2. In the 1800s, Priestley, Ingenhousz, and van Niel discovered oxygen evolution and that sunlight and water are essential.
3. In the 1900s, Calvin used radioactive tracers to discover the Calvin cycle, where CO2 is fixed into 3-carbon compounds in chloroplasts, using enzymes like RuBisCO.
4. C4 and CAM pathways were discovered as alternatives to the C3 Calvin cycle that improve efficiency in hot/dry conditions through additional CO2
This study investigated the effects of spermine NONOate (a nitric oxide donor), ATP, and sodium/potassium buffer environment on the thermal stability of human hemoglobin using circular dichroism spectroscopy. The main findings were:
1) Spermine NONOate decreased the unfolding temperature of hemoglobin irrespective of buffer composition.
2) ATP increased the unfolding temperature by 3°C in both sodium and potassium buffers.
3) The effects of ATP and nitric oxide depended strongly on the specific buffer ionic composition.
This document presents 5 theoretical chemistry problems. Problem 1 involves identifying compounds containing element X based on provided information about mass percentages and oxidation states. Key compounds identified are:
A is ClF. B and Z1 contain Cl, F, and O. Z1 is ClOF3.
The document also involves writing chemical equations, identifying possible isomers, explaining conductivity, and writing hydrolysis equations.
SIMONA CAVALU_Raman and Surface Enhanced Raman Spectroscopy of 2,2,5,5-Tetram...Simona Cavalu
ABSTRACT: 2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) labeled bovine
serum albumin and cytochrome c at different pH values were prepared and
investigated using Raman–resonance Raman (RR) spectroscopy and surface enhanced
Raman scattering (SERS) spectroscopy. The Raman spectra of tempyo labeled proteins
in the pH 6.7–11 range were compared to those of the corresponding free species. The
SERS spectra were interpreted in terms of the structural changes of the tempyo labeled
proteins adsorbed on the silver colloidal surface. The tempyo spin label was found to be
inactive in the Raman–RR and SERS spectra of the proteins. The a-helix conformation
was concluded to be more favorable as the SERS binding site of bovine serumalbumin.
In the cytochrome c the enhancement of the bands assigned to the porphyrin macrocycle
stretching mode allowed the supposition of the N-adsorption onto the colloidal surface.
This document discusses Raman and surface enhanced Raman spectroscopy (SERS) experiments performed on bovine serum albumin and cytochrome c proteins labeled with 2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) spin label at various pH values. The Raman and SERS spectra of the labeled proteins are compared to the unlabeled proteins. For bovine serum albumin, the SERS results suggest the α-helix conformation is favored at the binding site and the tempyo label does not significantly impact the vibrational structure. For cytochrome c, SERS enhancement of the porphyrin macrocycle bands indicates N-adsorption of
Mechanism of the Reaction of Plasma Albumin with Formaldehyde in Ethanol - Wa...IOSR Journals
The Spectrophotometric determination of the acid dissociation/ionisation constant (pKa) of plasma albumin-formaldehyde adduct in both water solution and Ethanol solutions was carried out in this study. The pKa values obtained in both media were used to establish the Bronsted-linear type constants from plots of pKa against logarithm of second order rate constants obtained at varying pHs in the study. The result of the pKa values obtained in both water solution and ethanol-water mixtures were found to be in the range of 5.0 - 8.0. This pointed to the fact that only lysine residue with pKa value 8.3 that might have possibly reacted with formaldehyde in this reaction of all the known amino acid residues in plasma albumin. The corresponding Brønsted-type plots proportionality constants (β) for the reaction in water and ethanol-water mixtures were found to be β = 0.059 and 0.0057 respectively. The reaction mechanisms that have low values for proportionality constants α or β are considered to have a transition state closely resembling the reactant with little proton transfer (Cox et al, 1988). Thus, one would suggest that the cross-linking of formaldehyde with plasma albumin in water and ethanol-water mixtures proceeds through little proton transfer
The document examines the effects of acid on the chlorophyll production and biomass of common duckweed (Lemna minor L.). Two experiments were conducted exposing duckweed to pH levels of 4.1, 5.4, and 6.5 (control) over 10-12 days. The first experiment showed no significant differences in biomass between pH treatments. The second experiment found significantly lower biomass at pH 4.1 compared to pH 5.4 and 6.5. Neither experiment found significant differences in chlorophyll content between pH treatments. The results partially supported the hypothesis, showing acid inhibited biomass but not through impacts on chlorophyll. Longer exposure periods or lower pH levels may be needed to impact chlorophyll.
Emergent ribozyme behaviors in oxychlorine brines indicate a unique niche for...Sérgio Sacani
Mars is a particularly attractive candidate among known astronomical objects
to potentially host life. Results from space exploration missions have provided
insights into Martian geochemistry that indicate oxychlorine species, particularly perchlorate, are ubiquitous features of the Martian geochemical landscape. Perchlorate presents potential obstacles for known forms of life due to
its toxicity. However, it can also provide potential benefits, such as producing
brines by deliquescence, like those thought to exist on present-day Mars. Here
we show perchlorate brines support folding and catalysis of functional RNAs,
while inactivating representative protein enzymes. Additionally, we show
perchlorate and other oxychlorine species enable ribozyme functions,
including homeostasis-like regulatory behavior and ribozyme-catalyzed
chlorination of organic molecules. We suggest nucleic acids are uniquely wellsuited to hypersaline Martian environments. Furthermore, Martian near- or
subsurface oxychlorine brines, and brines found in potential lifeforms, could
provide a unique niche for biomolecular evolution.
Probing the chemistries of flavin ring systems of p hydroxybenzoate hydroxyla...John Clarkson
J. Clarkson, B. Pulfey & P.R. Carey, “Probing the Chemistries of Flavin Ring Systems of p-Hydroxybenzoate Hydroxylase by Raman Difference Spectroscopy”, Biochemistry, 36, 12560-12566, 1997.
This document describes the synthesis of the allelochemicals heliannuols A and K, and the sesquiterpene helianane. It develops a regioselective method for cleaving a benzo-fused oxabicyclo(5.1.0)octane system under hydrogenation and radical conditions to generate the basic benzoxocane ring structure found in these compounds. Key steps include generating the gem-dimethyl group on 5-deoxy-heliannuol A through Bargellini alkylation, and performing a novel ring expansion through selective cleavage of a cyclopropane-annulated cycloheptane ring to form the 8-membered benzoxocane ring. This
April 1, 2016 Prof. Brian H. Lower Editor, ScienceBites.docxjustine1simpson78276
April 1, 2016
Prof. Brian H. Lower
Editor, ScienceBites
The Ohio State University
School of Environment & Natural Resources
210 Kottman Hall
2021 Coffey Road
Columbus, Ohio 43210 (USA)
Re: Revised Manuscript Version 2
Dear Dr. Lower,
Thank you for considering my revised manuscript for publication in ScienceBites, I am pleased
that you have requested minor modification of our manuscript. Below is a detailed point-by-point
explanation of how we addressed ALL of the reviewers’ comments in our revised
manuscript. To address these concerns we:
1. Modified and/or added additional text to the manuscript.
2. Modified Figure 1.
3. Added 9 new references.
4. Added 1 new co-author who performed additional work to address the concerns of
Reviewer #2 regarding the specificity of the polyclonal antibodies used in our
experiments.
We have also indicated the page and line number where new text can be found in our revised
manuscript. While these modifications increased the length of our manuscript, we attempted to
make text additions as brief as possible, while still addressing the concerns of both reviewers.
In addition, we moved our figure captions to a separate “Figure Legends” page in our manuscript
as you requested.
Should our revised manuscript be accepted for publication we request that our article appear the
environmental section of ScienceBites.
We appreciate the comments and suggestions of the two anonymous reviewers and the time and
effort of the editor. The input that we received from these people has definitely improved the
quality of our manuscript.
Best regards,
Susie A. Student
Reviewer 1 Comments:
1. The authors need to explain why the performed the AFM measurements in growth medium?
The use of growth medium adds complexity to the measurements as it contains a lot of proteins
and can increase the non-specificity interactions.
We agree with Reviewer #1’s comment that having proteins in the imaging fluid can add
complexity to the measurements. We regret that we mistakenly listed our imaging buffer as
“growth medium” in our original manuscript when we actually performed our AFM measurements
in phosphate buffered saline (PBS) at pH 7.4. We thank the reviewer for her/his careful attention
to detail and we have corrected our mistake in the revised manuscript. These changes can be
found on page 4, lines 11-13. The sentence now reads: “We performed Ig-RFM in phosphate
buffered saline (PBS), pH 7.4, on living S. oneidensis MR-1 cells that were deposited on a
hydrophobic glass cover slip using an Asylum Research MFP-3D-BIO AFM or a Digital
Instruments Bioscope AFM (15, 16).
2. The authors need to comment on the stability of the bacteria during AFM measurements.
Bacteria were only deposited on glass and that allows the cell to move under liquid.
We apologize for not making this point clear in the original manuscript. For the experiments
show.
Similar to J. biol. chem. 1986-fushitani-8414-23 (20)
April 1, 2016 Prof. Brian H. Lower Editor, ScienceBites.docx
J. biol. chem. 1986-fushitani-8414-23
1. THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 261, No. 18, Issue of June 25, pp. 8414-8423 1986
0 1986 by The American Society of Biological Chemists, Inc. Printed in L k A .
Oxygenation Properties of Hemoglobin from the Earthworm,
Lumbricus terrestris
EFFECTS OF pH, SALTS, AND TEMPERATURE*
(Received for publication, December 16,1985)
Kenzo FushitaniSBT,
Kiyohiro ImaiB, and Austen F. RiggslI))
From the *Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka
560,
Japan, the $Department of Physicochemical Physwlogy, Medical School, Osaka University,Nakanoshima, Osaka 530, Japan,
and the TDepartment of Zoology, University of Texas, Austin, Texas 78712
Oxygen equilibrium curves of the extracellular chains of vertebrate hemoglobins (5). The whole moleculehas
hemoglobin from Lumbricus terrestris weredeter- been reconstituted from dissociation products and theshape
mined under a variety of conditions. These data were has been compared with that of the native molecule by scan-
characterized by (i)a rather small free energy of coop- ning transmission electron microscopy (6). Oxygen-binding
erativity (1.6-2.8 kcal/mol), (ii) a large and strongly properties of L. terrestris hemoglobin have been studied by
pH-dependent Hill coefficient with a maximum value several investigators (7-9). Giardina et al. (10) measured
of 7 9 (iii)a high sensitivity of the upper asymptote of
., oxygen binding by earthworm’ hemoglobin under a variety of
the Hill plot to pH, and (iv) a maximum association conditions and showed that the Hill coefficient, n, depends
constant as large as that of the free subunit of human
# I
strongly on pH and hasa maximum value of 4 at pH 7.8; the
hemoglobin A. oxygen affinity varies between 0.4-7 mm Hg between pH 5.5
The effects of LiCl, KCl, NaCl, BaCl,, CaC12, SrC12,
and 10. Vinogradov et al. (11)have reported similar values for
and MgClz on the oxygen equilibrium were measured.
Cations, not C1-, were found to control oxygen binding. hemoglobin from L. terrestris.
Divalent cations have a larger effect on oxygen affinity Recently, Weber (12) showed that cations control the oxy-
than monovalent cations, and their effectiveness de- gen affinity of the extracellular hemoglobin of Arenicolu mar-
creased in the order listed above within each valence ina. Addition of cations such asNa+ or Mg2+ enhanced coop-
class. These specific effects depend in part on ionic erativity and raised the oxygen affinity by binding to hemo-
radiusandcannot be explainedinterms of ionic globin at high levels of oxygenation. He also showed that
strength.Thedataindicatethatthe oxygenation- protons lower the oxygen affinity by preferential binding to
linked binding of a Ca2+ ion is accompanied by the hemoglobin a t high saturation levels. The mechanisms of
release of two protons; the binding of a Na+ ion is cationic and protonic interaction in theseextracellular hemo-
associated with the release of one proton. These find- globins must be quite different from those in human hemo-
ings indicatethat theoxygenation-linked cation-bind- globin A, where anions and protons decrease the oxygen
ing site contains two acid groups that do not readily affinity by preferential binding mainly to molecules in the
dissociate their protons except when replaced by cat- low affinity state (13, 14). The important study by Santucci
ions. et al. (15) has shown for hemoglobin of Octolasium complun-
Incubation at either pH 6.2 or 8 9 had no effect on
. atum that oxygen binding becomes independent of pH be-
subsequent measurements of oxygen equilibria at pH tween pH 7 and 8.5 at sufficiently low cation concentrations.
7.8. The apparent heat oxygenation was found to be
of The pH dependence of cooperativity and cationic control of
-1 1.8, - . ,and - . kcal/mol at pH 9 0 7 4 and 6.6,
73 93 .,., oxygen affinity has also been found in several other extracel-
respectively. These differences indicate that proton- lular hemoglobins of annelids (16-23).
binding processes contribute to the heat of oxygena- In the present study we have measured oxygen binding
tion. between 1 and 99% saturation with high precision under a
wide variety of conditions which includes different kinds and
concentrations of salts, changes inpH,temperature,and
The extracellular hemoglobin of the earthworm, Lumbricus protein concentration.
terrestris, consists of 12 subunits, arranged as two superim-
posed hexagonal disks (3), about 30 nm in diameter and 20 EXPERIMENTALPROCEDURES
nm high (4)with a molecular weight of 3-4 X lo6. Although Preparation of Hemoglobin-Earthworms, originally obtained in
its chain composition and the spatial arrangement of the Ontario, were purchased from the Wholesale Bait Co., Hamilton, OH
chains have not yet been determined, one of the polypeptide 45015. They were cut with scissors at a position just anterior to their
chains has been sequenced and shown to be homologous to hearts and bled into CO-saturated 0.1 M sodium phosphate buffer
(pH 7.0) containing 3 m phenylmethylsulfonyl fluoride as a protease
M
* This work was supported by National Science Foundation Grants inhibitor. The crude solution was centrifuged to remove cellular
PCM 8202760 and DMB-8502857, Welch Foundation Grant F-213, matter. The hemoglobin was precipitated by adding polyethylene
and National Institutes of Health Grant GM28410. A preliminary glycol (8,000 average molecular weight, Sigma) to a concentration of
account of some of this work has been presented (1,2). Thecosts of
publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “aduer- Originally identified in 1975 as Lumbricus sp. (10) and recently
tisement” in accordance with 18U.S.C. Section 1734 solely to indicate reidentified (1983) asOctohium complnmtum (6) although the
this fact. worms used by Giardina et al. (10) were referred to as L. terrestris in
11 To whom reprint requests should be addressed. 1984 (15).
8414
2. Oxygenation
Properties of Lumbricus terrestris
Hemoglobin 8415
10%.The precipitate was redissolved in 0.05 M Tris-HC1, pH 8.0, and Analysis-Oxygenation data were expressed in terms of the Hill
twice pelleted a t 40,000 rpm for 2 h. Pellets were resuspended in this plot (log(Y/(l- Y ) )uersus log P ) where Y is fractional saturation of
CO-saturated buffer and stored in liquid nitrogen. For oxygenation the hemoglobin with oxygen and P is the oxygen pressure in mm Hg.
experiments, an aliquot of the frozen hemoglobin sample was thawed, Overall oxygen affinity and cooperativity were characterized in terms
centrifuged a t low speed, and subjected to gel chromatography on a of oxygen pressure a t half-saturation (PW) maximum slope of the
and
Sepharose CL-GB column equilibrated with 0.1 M sodium phosphate Hill plot (hex) or slope of the Hill plot at half-saturation (nw).
buffer (pH 7.7) containing 0.1 M NaCl saturated with CO. Less than Cooperativity was also expressed in terms of a free energy change
5% of the total hemoglobin was found to be dissociated into %z defined as AG, = R T ln(K,,,/KJ where K,,, and Kl are estimated
subunits during the freeze-thaw treatment on the basis of gel chro- association constants for the last and first oxygens bound to hemo-
matography. The eluted fractions were concentrated to about 3.2% globin. Values of n were determined by plotting values of n against
,
w/vby ultrafiltration (Toyo Roshi apparatus, UM-10 membrane). logp. Here, n is the slope of the line connecting two adjacent points
The concentrated hemoglobin solutions were dialyzed against 0.05 M on the Hill plot. The apparentassociation constants for the binding
Tris-HC1 (pH 7.0 a t 25 "C) containing 0.1 M NaCl and stored on ice of the first and last oxygens to hemoglobin were estimated approxi-
as the CO form. mately with an m-step Adair's oxygenation scheme (28),
Oxygenation Measurements-Oxygen equilibria were measured
with an improved version of an automatic oxygenation apparatus (24) log(Y/(l - Y ) )= log P + log Kl at P o -+ (1)
without the use of an enzymatic reducing system. Deoxygenation log(Y/(l - Y ) )= log P + log K,,, at P -+ m (2)
and/or reoxygenation data were acquired in real time by a model
PDP-ll/vOB microcomputer (Digital Equipment Corp., Maynard, where Kl and K,,, are apparent association equilibrium constants for
MA) and were stored on floppy disks. The absorbance value corre- the first and lastoxygens.
sponding to 100%saturation with oxygen was obtained by extrapo- The magnitude of the Bohr effect was estimated by the following
lating a &4 versus 1/P plot to 1/P = 0 where A.4 is the absorbance expression (29).
change upon oxygenation and P is the partial pressure of oxygen (13).
The buffer was 0.05 M BisTris/propane' over the range pH 6.2-9.0. h = -Alog Pw/ApH (3)
The pH adjusted with concentrated HCl at the
was same temperatures
Here, h is the number of protons released by Hb/oxygen bound.
as those used in the oxygenation experiments. The hemoglobin con-
Similarly the magnitude of the effect of a given ion on oxygenation
centration was 60 F M on a heme basis, unless otherwise stated. The is expressed by
bound CO was removed from the hemoglobin by light while the
sample was flushed with pure 0 in a rotating flask immersed in ice
' I = +Alog PW/Alog[ion] (4)
water just prior to the measurements.
Reproducibility of the Oxygenation Curve-Reproducibility of the where I is the number of ions released by Hb/oxygen bound.
oxygen equilibria is affected by two factors: the stability of the The apparent enthalpy of oxygenation, A H , was calculated from
automatic oxygenation apparatus and that the hemoglobin sample.
of the slope of the plot of log PW versus 1/T between temperatures of
Careful inspection of the data showed that oxygenation curves meas- 283 and 303 K.
ured under the same conditions within a few days of one another The Monod-Wyman-Changeux (MWC) allosteric model (30) de-
could be superimposed over the whole saturation range between 1 scribes the oxygenation of hemoglobin as,
and 99% with a variance of 1-2% of P W and n - values where Pw is
,
the oxygen pressure a t half-saturation and n is the maximum value
,
- Y=
+
L K T P ( ~ KTP)""' +
+ K ~ p ( 1 KRP)""' (5)
of the Hill coefficient. Therefore, the reproducibility of the oxygena- L(1 + KTP)" + ( 1 + KRP)'"
tion curve was found to depend mainly on irreversible alteration of where rn is the number of interacting binding sites for oxygen, KT
the hemoglobin sample during storage such as autoxidation to a and K are theintrinsic association equilibrium constants for oxygen
R
hemichrome. The PW value dropped by 3%, andn decreased from
- binding to the T state and the R state, respectively, and L is the
5.0 to 4.8 at pH 8.9 after 3 weeks. Purified CO-hemoglobin samples allosteric constant. Recently, Decker et al. (31) introduced a conven-
gave a variance of about 6% for PW values and 7% for n.-= values ient graphic method for analyzing oxygenation data on the basis of
after storage on ice for 3 months. the MWC model. Imai and Yoshikawa (28) modified their formula
Estimation of Methemoglobin Content-No complete set of absorp- slightly, as follows.
tion coefficients for estimating methemoglobin content over the pH
range of 6.2-8.9 is available for this hemoglobin. We failed to deter- log Z = ( m - 1) log X + log L (6)
mine the coefficients for methemoglobin because, as Ascoli et al. (25)
reported, the spectrum of the L. terrestris hemoglobin changes upon Here, 2 = (KR - Q ) / ( Q - KT), Q = Y/((l - Y)P), and X = (1 +
oxidation from that of typical aquomethemoglobin to that of hemi- +
K T P ) / ( ~ KRP). Z is the ratio of the difference in affinity between
chrome. The millimolar extinction coefficient for oxyhemoglobin at hemoglobin in the R state and hemoglobin a t a given value of P to
576 nm, obtained by the pyridine hemochromogen method, was 16.4, that between hemoglobin at the given value of P and the T state. X
which is close to the value of 14.6 a t 577 nm for the oxy form of is the ratio of the binding polynomial for oxygenation of the T state
human HbA (26). We used the value of34.4 for the millimolar to that for oxygenation of the R state. When KT and KR values are
extinction coefficient of the pyridine hemochromogen a t 557 nm. already known, log Z can be plotted against log X,yielding a straight
Methemoglobin content was estimated by using spectra taken before line. The values of m and log L can then be determined from the
and after oxygenation measurements. The ratio of absorbance a t a slope and the intercept on the ordinate at log X = 0. In the present
trough near 505 nm to that ata peak at 576 nm provides an index of study, we used Kl andK,,, for KTand KR,respectively.
methemoglobin. The value of this index for hemoglobin solutions We also attempted to estimate the number of interacting binding
between pH 7.0-7.8 before measurements was 2.99 0.03 S.D. (26 sites, m,by using Kegeles' expression (321, m = n,-/y-, derived
measurements). The index for human HbA obtained under similar from the MWC model. However, the slope is too steep to permit
conditions was 2.99 f 0.06 S.D. (10 measurements). The value of2.99 accurate values to be obtained.
for human HbA corresponded to 2.8% f 1.1 S.D. methemoglobin
(27). We estimated that 2-4% methemoglobin was present in the RESULTS
purified stock solutions of L. terrestris hemoglobin by these approxi-
mate criteria. Methemoglobin content after oxygen equilibrium meas- Oxygen-binding Properties: Effectof p H and Calcium-Hill
urements was usually estimated to be 6-8% and never more than plots of oxygen equilibria of L. terrestris hemoglobin are given
10% except for three experiments carried out atheme concentrations in Fig. 1. Measurements were made between pH 6.2 and 8.9
under 20 pM (see Fig. 7). Estimation of MetHb content at other pH using 0.05 M BisTris/propane with and without different
values was similar. The index decreased to 2.90 f 0.03 S.D. before additional salts. Values of Pw and n obtained from these
-
measurement 3 months afterpurification. curves are listed in Table I together with other parameters
and areplotted against pH in Fig. 2. Fig. 2 also includes data
'The abbreviations used are: BisTris, 2-[bis(2-hydroxyethyl)- from human hemoglobin A (34) for comparison. The datafor
MetHb, methemoglo-
amino]-2-(hydroxymethyl)-propane-1,3-diol; L. terrestris hemoglobin in Figs. 1 and 2 have four major
bin; MWC, Monod-Wyman-Changeux. features as described in the following paragraphs.
3. 8416 Oxygenation Properties of Lumbricus terrestris Hemoglobin
log P
FIG.1. Hill plots of oxygen binding by hemoglobin of L terrestris at different pH values and salt
.
concentrations. Symbols: Y,fractional saturation of hemoglobin with oxygen; P, partial pressure of oxygen in
mm Hg. Conditions: 50 mM BisTris/propane, 3 m NaCl, 25 "C, with various amounts of additional salts;
M
hemoglobin concentration, 60 pM heme. A, no additional salt. pH from the left to right: 8.56,8.13, 7.75, 7.36, 7.00,
6.58, 6.23. B, additional salt, 0.1 M NaCl. pH from the left to right: 8.86, 8.52, 8.10,7.75, 7.35, 6.95, 6.55, 6.16. C,
additional salt, 0.1 M NaC1,25 mM CaC12. pH from the left to right: 8.88,8.48,8.10, 7.73,7.36,6.98,6.58, 6.20. The
solid straight lines with a slope of unity indicate the lowest lower and highest upper asymptote of the Hill plots.
The lowest lowerasymptote was obtained from cunres in A, the highest upper asymptote from C. Their intercepts
on the ordinate at log P = 0 give log K L =i -1.6 (KL 0.024 mm Hg") and log KH = 0.58 (KH 3.8 mm Hg-'),
= =
where K L and KH are the lowest and highest values, covered by the present experiments, corresponding to the
association equilibrium constant for the low oxygen affinity state (thelowest first Adair constant, KJ and that for
the high oxygen affinity (the highest last Adair constant, Km), respectively. The asymptotes for free B subunit and
hemoglobin M Milwaukee corresponding to 4 and 0.004 mm Hg", respectively, are also shown for comparison
(33).
1)The shape of the oxygen equilibrium curve varies greatly ative binding of oxygen are smaller than those of human HbA
with pH. In the absence of calcium, the value of kx rises (33), although the values of the Hill coefficient are much
from 2.5-3.0 at pH6.2-6.6 to a maximum of 6.5-7.2 near pH larger (Fig. 2and Table I). Thelowest valuefor Kl, 0.024 mm
8.1 and decreases to 5 at pH 8.9. The value of nmlu rises to a Hg-l, was obtained in the absence of added salt (Fig. l )A.
maximum value of 7.9 at pH7.5 in the presence of calcium; The highest value for K,, 3.8 mm Hg-l, was obtained in the
values of n at both extremes of pH remain unchanged.
- presence of 0.1 M NaC1, 25 m CaClz (Fig. lc, Table I). The
M
2) Salts increase overalloxygen affinity. This effect in- asymptotes corresponding to these values are shown in Fig.
creases with pH and is more pronounced with CaClz than 1. Values of free energy of cooperativity (AG,) are listed in
NaC1. The pH of the maximum Bohr effect is decreased by Table I. The pH dependence ofAG, is similar to that of hax
salts. The maximum valuesof the Bohr coefficient (Alog Ps0/ (Fig. 2).The maximum AG, value, 2900 cal/mol, was obtained
ApH) are -0.35 (pH 8.3-8.5), -0.53 (pH 8.6-8.9), and -0.77 in the presence of 0.1 M NaCl, pH 8.52. This value is about
(pH 7.4) in the presence of no additional salt, 0.1 M NaCl, 80% of the maximum value,3640 cal/mol, for human HbA in
and 0.1 M NaC1, 25 m CaC12,respectively.
M 0.1 M Cl-, 2 m 2,3-diphosphoglycerate,pH 7.4 (33).
M
3) The two asymptotes of the Hill plot depend on pH Effect of Different Salts on Oxygenation-Fig. 4 and Table
differently. The values of Kl and K , calculated from the I1 show the effects of chloride salts of Li+, Na+, K+, Mf,
extrapolated asymptotes are listed in Table I. Their pH de- Ca2+,and of mixtures at pH 7.36, 25 "C. Monovalent salts
pendence is given in Fig. 3. The value of Kl is about 0.024 have almost no effect on nmaror log Ps0 up to 0.1 M, and
mmHg" between pH 6.2 and 8.2 in the absence of added changes inthe Hill coefficient are negligible up to 1 M.
salt. However, the value of K , increases from 0.43 mm Hg-l However, the oxygen affinity doubles between concentra-
salt
at pH 6.2 to 1.9 mm Hg-I at pH 8.6. NaCl and CaClz both tions of 0.1 and 1M.The effect of divalent salts is pronounced
enhance the pH dependence of K,. In the presence of CaCh at much lower concentrations; the effect of 0.1 M CaC1, is
(Table I, C), the value of K, reaches a plateau near pH 8 similar to thatof 1M NaC1. The combination of Ca2+and 0.1
corresponding to a value of3.8mmHg". The value of Kl M NaCl causes a concentration dependence of log Pm similar
increases only slightly with pH in thepresence of Ca2+. to that caused byCa" alone, whereas ha= a quite
shows
4) The overall free energychanges associated with cooper- different dependence on CaClz compared NaC1. with
4. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8417
I I I
TABLE I 9
i
Values of oxygenation parameters for hemoglobin of L. terrestris
obtained under differentconditions of salt and pH
In 0 0 M BisTris/propane/HCl buffer, at 25 "C; hemoglobin con-
.5
centration 60 PM on a heme basis. A, no additional salt; B, in the c
E
presence of added 0.1 M NaCI; C, in the presence of added 25 m M
CaCL 0.1 M NaCl. ----""""
A
8.56
mm Hg
5.9 6.3 5.4
eallmol
2400 11.2 71 8.9
'
TM T
8.13 8.2 6.5 5.6 2400 12.4 72 9.0
7.75 10.3 6.0 5.2 2300 11.6 72 8.3
7.36 12.7 6.6 5.1 2300 1.
11 70 9.4
7.00 15.2 4.1 3.6 2200 8.5 67 6.1
6.58 16.4 3.1 3.0 1700 6.5 75 4.1
6.23 17.1 2.5 2.3 1700 5.4 63 4.0
B
8.86 2.7 5.0 4.9 2300 9.9 46 10.9
8.52 4.1 6.9 6.6 2900 10.8 61 11.3
8.10 6.3 7.5 6.5 2800 10.8 69 10.9
7.75 9.0 7.2 5.6 2600 10.8 74 9.7
7.35 12.1 6.2 5.3 2400 11.1 73 8.5
6.95 15.4 4.5 3.5 2100 8.4 66 6.8
6.55 17.0 3.0 2.5 1700 6.5 67 4.5 FIG. 2. The effect of pH on overall oxygen affinity (log P)
,
6.16 17.3 2.7 2.2 1700 5.1 72 3.8 and cooperativity (%=) at 25 OC. Symbols: Pw, partial oxygen
pressure at half-saturation; hx, maximal slope of the Hill plot.
C Values of these parameters were obtained from the Hill plots in Fig.
8.88 1.6 4.9 4.9 2500 8.0 57 8.6 1.A, no salt added; 0 , O . l M NaCI; 0 , 2 5 m CaC&with 0.1 M NaC1.
M
8.48 1.9 5.8 5.5 2600 9.4 56 10.4 Broken lines show a plot of log P , and for human HbA in 0.1 M
8.10 2.6 6.8 6.4 2800 10.5 61 11.1 NaCl(34). P,,, is the median oxygen pressure.
7.73 3.9 7.9 6.9 2800 12.4 67 11.8
I ' 1 I (
7.36 6.9 7.7 7.1 2600 11.9 67 11.5
6.98 11.6 5.1 4.2 2100 9.7 70 7.3
6.58 15.5 3.1 2.7 1900 6.2 71 4.4
6.20 17.2 2.6 2.3 1700 5.7 70 3.7
a Oxygen pressure at half-saturation.
* Maximum slope in Hill plot.
Slope at half-saturation in Hill plot.
Free energy of interaction; AG, = RTln(Km/Kl).
e Estimated number of interacting sites based on the
linearized plot
(Equation 6).
f Oxygen saturation giving a n -
,.
Estimated interacting sites based on Kegeles' analysis.
Oxygenation in the presence of 3 m NaC1, 25 m CaCli
M M -2.oL
" .
(Fig. 5) shows that CaClz increases the oxygen affinity and 8 I
cooperativity by shifting the upper asymptote to the left. 60
. 7.O 80
. 9.0
PH
Although 25 m CaClz has no effect on the lower asymptote,
M
125 m CaClz causes a significant shift to the left which is
M FIG.3. The effect of pH on K1and K. Kl and K,,, the
, are
association equilibrium constants for the first and last
oxygens to be
associated with a decrease in ha. Similar effects were ob- bound. Values of these parameters were obtained from the Hill plots
served with MgClz. in Fig. 1. A, no salt added, 0,O.l M NaCI; 0 , 2 5 mM CaClz with 0.1
The effect of 25 m S?+ and Ba" was also examined at
M M NaCI; 0, estimated values of Kl andK, obtained by extrapolation
pH 7.37. Positions of the lower asymptote of the oxygenation to zero MetHb of the data obtained with partidy oxidized hemoglobin
curves were similar to those for Ca" and M$+ whereas the (see t x )
et.
position of the upper asymptote differed. The log Pmvalues
were: 0.98,0.86, 0.80, and 0.74 for M$+, Sr2+,Ca", and Ba2+, the Hill coefficient by measuring oxygen binding by hemoglo-
respectively. Thus, Ba2+with the largest ionic radius was the bin which had been partially oxidized with potassium ferri-
most effective and M$+, the smallest ion, had the least effect cyanide at pH 7.3 (Fig. 6). The ferricyanide was assumed to
among the divalent cations so far examined, and Sr2+ and react completely with heme. Methemoglobin content up to
ea2+with intermediate ionic radii had effects between those 30% had no signifkant effect on the P m value which was
of Ba" and M$+. Although the difference between the log 11.2-11.8mm Hg (average, 11.5mm Hg k 0.2 S.D.). The
P m values for Sr2+ and Ca" or Ca" and Ba2+are only 0 0 .6 value increased slightly at 50% methemoglobin. However the
this is 15% in the value and appears to be well beyond the
P value decreased with an increase in methemoglobin (6%
estimated experimental error. Inositol hexaphosphate had no decrease with 10% methemoglobin). We estimated values of
effect on oxygenation; the data obtained with 2 m inositol
M both Kl and K , at 0% methemoglobin byextrapolating plots
hexaphosphate at pH 7.41 could be superimposed on those of log Kl or log K , against MetHb content. The extrapolated
obtained in itsabsence. values are -1.7 and 0.13 for log K1 and log K,, respectively
Effect of Partial Oxidation-We examined the effectof (Fig. 3). These results indicate that Kl and K,,, were not
partial formation of methemoglobin on oxygen affinity and affected significantly by the methemoglobin present under
5. 8418
c
0
:m
I
T
Oxygenation Properties of Lumbricus terrestris Hemoglobin
T
cg ~~ I 25°C
Q!
-
0
0
0'50 10 30 50
% Meihernoglobin
Salt Concentration Hb Concentration (pM heme)
FIG. 4. Effect of salts on overall oxygen affinity (log Pso) FIG. 6 (left). Effect of partial oxidation on overall oxygen
and cooperativity (n-). Symbols: PSO, partial pressure of oxygen affinity (log Ps0)and cooperativity (ram-). Oxygenation curves
at half-saturation;n- maximum slope of Hill plot. Hemoglobin
,, the were measured i 0.05 M BisTris/propane/HCl, 0.1 M NaCl, pH7.30
n
concentration, 60p~ on a heme basis, "C.A, effect of monovalent
25 at 25 "C. Hemoglobin concentration, 60 ptM on a heme basis. The
cations: 0, LiCl (pH 7.33-7.36);0,NaCl (pH 7.38-7.39); A, KC1 (pH abscissa showsthe percentage of methemoglobin.
7.36-7.40). B, effect of divalent cations:0, MgClz in the presence of FIG. 7 (right). Effect of hemoglobin concentration on overall
0.1 M NaCl (pH 7.34-7.38); 0, CaClz in the presence of 0.1 M NaCl oxygen affinity (log P60)and cooperativity(k-). Oxygenation
(pH 7.36-7.38); A, CaC12 only (pH 7.36-7.40). Broken lines show curvesweremeasured i 0.05 M BisTris/propane/HClbuffer,pH
n
effect of monovalentsalts; data from A included for comparison. 7.38-7.39, at a concentration of hemoglobin between and 600 p~
0.6
on a hemebasis.
3t 1 TABLE I1
Effect of salts on overall oxygen affinity and number cations taken
of
up uponoxygen binding
In 0.05 M BisTris/propane/HClbuffer at 25"C; pH7.34-7.40;
hemoglobin concentration,60 NM heme.
Cation
Salt"
bound/O.
mM
0.75 580 Li+
940 Na' 0.52
K+ 0.45 820
+
M$+ 0.1 M Na+ 110 0.33
Ca2+ 0.37 27
0
+
Ca2+ 0.1 M Na+
0.37 41
-3- * Salt concentration neededt o double overall oxygenaffinity.
- 1 0 1 2 3
log P
not required to explain the very small shifts.
FIG. 5. Effect of calcium chloride on oxygen binding by Reversibility ofOxygen Equilibria with Changes of pH-
hemoglobin of L terrestris in terms of the Hill plot. Buffer:
.
0.05 M BisTris/propane/HCl and 3 M NaCl (pH 7.36-7.40) at 25 "C.
m Giardina et al. (10) reported that the shape of the oxygen
Hemoglobin concentration, 60 W M (heme basis). P, oxygen pressure, equilibrium curve of hemoglobin incubated at either pH 6.0
mmHg; Y , fractional oxygen saturation. Ca2+ concentration: from or 10 for 1 h is modified irreversibly. Several workers have
the left to right, 125, 25,0 mM. reported similar phenomena for other extracellular annelid
hemoglobins (35-37). We have re-examined this property with
the conditions used. L. terrestris hemoglobin. Oxyhemoglobin solutions at pH6.2,
Effect of Hemoglobin Concentration-Fig. 7 shows the effect 7.8, and 8.9 were prepared as described under "Experimental
of increasing the hemoglobin concentration from 0.6 to 600 Procedures." Each solution was incubated for 1 h at 25 "C
pM at pH 7.57 in the presence of 0.1 M NaC1. Values of log and passed through a Sephadex G-25 column (pH 7.8) so that
P50 nmax not of high accuracy at concentrations of 2
and are the three samples had the same fiial pH (Fig. 8, Table 111).
p~ or lower because of significant autoxidation. The 100-fold The plots at the in Fig. 8 show the Hill plots for a control
left
decrease in concentration from 600 to 6 PM is associated with experiment, where the curves for the hemoglobin samples
an increase in Pw of about 7% and a decrease in nmarfrom 6 untreated and treated(i.e. incubated and passed through the
to 5. The upper and lower asymptotes of the Hill plots shift column) at pH 7.8 are superimposed. Likewise, the plots at
only slightly upon the dilution of hemoglobin sample. The the right in Fig. 8 show five curves. Two of these show data
small changes in log PSoand at or below 20 PM heme are obtained at pH 6,2 or 8.9 and not brought back to pH 7.8.
very closely correlated with methemoglobin formation; the Three curves show data on samples treated at pH6.2, 7.8, or
P0 'value is linearly related to the MetHb index with. a
5 8.9 and brought back to pH 7.8. Over a range of 1-99%
correlation coefficient of 0.97. The MetHb index is invariant saturation, their agreement is excellent. It was found that
between 60 and 600 pM heme. Although a small degree of addition of 25 m CaClz to a solution incubated at pH 8.9
M
subunit dissociation may accompany dilution, dissociation is improved the reversibility even further for pH changes from
6. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8419
I I I -
n
0 8 *A
pH 8 9-7.8
. 0 .E
FIG. 8. Test of the reproducibility
of oxygenation after changing the
buffer. Hemoglobin solutions incubated
for 1 h at each pH (untreated; shown
with open symbols) were brought back to
the same pH 7.8 with Sephadex G-25
(treated; shown with solid symbols). Left
two curves are shown for control pur-
poses.
0
-2 1
-I 0
I t I I I I
-I 0 I 2 -I 0 I 2
log P
TABLE I11 TABLE IV
Reversibility of oxygenation parameters upon changing pH of Apparent heat of oxygenation (kcal/mol)
hemoglobin solution In 50 m BisTris/propane/HCl buffer with 0.1 M NaC1. Hemoglo-
M
In 0.05 M BisTris/propane/HCl buffer with 0.1 M NaCl at 25 "C. bin concentration, 60 pM heme. Heats of oxgen binding at oxygen
Hemoglobin concentration, 60 pM heme. Buffer was changed with saturations of 50%. Values were corrected for heat of solution of
Sephadex G-25. P50, oxygen pressure at half-saturation; n-, maxi- oxygen (3 kcal/mol).
mum slope in Hill plot. pH L. terrestris Human H b
Condition" PO
S n,
- MetHb indexb 9.0-9.1 -11.8 -15.3"
7.8 (untreated)" 6.7 7.64
2.85 2.99 -9.3 7.4 -7.5
2.67 72.81 . 8
.h7 6.0 7.59 -11.2
6.5-6.6 -9.3
5.9 7.83 6.2-7.8 In 50 mM BisTris or Tris/HCl buffer with 0.1 M NaC1, at pH6.5,
4 2.80 5.9 7.87
8.9+7.gd 7.4, 9.1 (27).
8.9-7.8' 7.83 5.1
2.42 2.95
Left column shows starting conditions. Right column shows con-
pH dependence of n,,, are qualitatively in agreement with
ditions after change (see legend for Fig. 8).
results obtained by Giardina et al. (lo), Santucci et al. ( E ) ,
Ratio of absorption at minimum value near 505 nm to that at the
and Vinogradov et al. (ll),
a peak in oxy form. Left, before measurement; right, after measure- although our n values are consist-
ment. ently higher evenwhenexpressed as nsOrather than n-
No buffer change with Sephadex G-25. (Table I). Qualitatively similar properties have beenreported
Incubated at pH 8.9 with 25 m CaC12.
M for extracellular hemoglobins from other annelids, both po-
e Incubated at pH 8.9 without 25 m CaC12.
M lychaetes and oligochaetes: Amphitrite ornata (20), Lumbri-
nereis tertraura (19), Pheretima hilgedorfi (22), and Eiseniu
8.9 to 7.8. The numerical data for these experiments (Table foetida (23).
111) show excellent reversibility with respect to pHchange. This pH dependence has been suggested to result from a
Effect of Temperature on Oxygenation-Oxygen equilibrium greater sensitivity of the upper asymptote to pH than the
curves were determined at 10,15, 20,25, and 30 "C at pH 6.6, lower one (20). Recently, Weber (12) clearly showed for A.
7.4, and 9.0 in the presence of 0.1 M NaC1, but satisfactory marina hemoglobin that theupper asymptote in theHill plot
data at pH 9.0 and 30 "C could not be obtained because of shifted to the left with an increase in pH but that the lower
significant autoxidation. The dependence of oxygenation of asymptote hardly moved. He suggested that theoxygen affin-
L. terrestris hemoglobin on temperature is similar in extent ity of the high affinity states depends on cation and proton
to that for human HbA (38). Overall heat of oxygenation binding. Our data (see Fig. 3) for L. terrestris hemoglobin are
(AI&) was obtained by plotting log Pm against 1/T. The completely consistent with this picture. The upper asymptote
values are listed in Table IV, together with data for human is more sensitive to pH than the lower one although the lower
HbA (27). asymptote does shift slightly above pH 8.0 in the absence of
salts andgradually shifts to theright in thepresence of 0.1 M
DISCUSSION
NaCl and 25 m CaClz abovepH 7.0.
M
Oxygenation Characterization of L. terrestris Hemoglobin- The data in Fig. 3 may be interpreted in terms of at least
Oxygenation parameters for L. terrestris hemoglobin in the two high affinity statesin L. terrestris hemoglobin.One
present work are compared in Table V with those for the appears at low pH independent of the presence of salts (KmL
same hemoglobin obtained by other workers, for hemoglobin = 0.43 mm Hg-') and the other at high pH and/or high salt
from two related earthworms, for chlorocruorin from Potam- concentrations (K," = 3 8 mm Hg-l, consequently equal to
.
ilk leptochaeta, and for human HbA. Our data on the strong KH). Free energy differences between KL and KmL,and K,"
7. 8420 Oxygenation Properties of Lumbricus terrestris Hemoglobin
TABLE V
Oxygenationparameters for extracellular hemglobin and chloroeruorin
Temperature
Source" pH P, n Bohr effect AHb Reference
oc
L. terrestris 25 6.2-9.0 1.63-17.3 2.5-7.9 -0.35-0.77 -7.5 to -11.8
7 7.3 2 -8.0
20 7.3 a (3.4)'
10 7.12 3.5 (3.0) -0.25
10 7.21 4.8 (2.3)
15 7.10 2.88 5.21 -0.4
15 7.44 3.89 5.20
15 7.10 4.98 5.13
25 7.70 5.28 5.41 -0.4 -10.2
25 7.44 6.78 5.30 -9.1
25 7.10 9.20 5.11 -10.6
22 6-9 (2.7-15.8) (1.8-5.0) (-0.54)
0. complnnatom 20 5.4-10.0 (2.2-6.9) (1.6-4.2) (-0.64) -6 to -13.7
E. foetida 20 5.1-9.4 (0.59-4.4) (1.8-3.5) -0.44
P.leptochaeta 25 6.2-9.2 11-420 1.14-5.82 -0.98 -3.9
Human HbA 25 6.0-9.0 161
.-6 2.53-2.98 -0.53 -11.2 to -15.3
'P. leptochaeta is the source of chlorocruorin; the othersare thoseof hemoglobin.
Values have been corrected for the heat of solution of oxygen, 3.0 kcal/mol, except for those from Ref. 9.
-
Values in parentheses areobtained bv reading or recalculating values from published data.
See Footn>te 1.
and KmH 1700 and 1300 cal/mol, respectively. The change
are a strongereffect than M$+ in E. foetida (23) and L. terrestris
of free energy of cooperativity with pH (Table I) is essentially (present data), but in ornata (20) the opposite is true.
A.
parallel to that of n- under three sets of conditions. Thus, Similar effects of salt concentration on log P 0 and
5
the pHdependence of the shape of the oxygen-binding curve (Fig. 4B) have been found in several other extracellular hemo-
between pH 6.2-9.0 results from the relative movements of globins (11, 15, 18, 23). One interpretation is as follows: (i)
the lower and upper asymptotes. The increase in at low cations bind to the liganded form of hemoglobin more than
pH appears to be due exclusively to an increase in the K,,, to the unliganded form at relatively low salt concentrations,
value with constant Kl) and the decrease in n,, at alkaline resulting in a shift of upper asymptote leftward (Fig. 5); (ii)
pH is due to a slight increase in K1 with the upper asymptote changes of n- value with cation concentration are due to
remaining unchanged. On the same basis, the shift of pH relative movements of both the upper and lower asymptotes.
giving the maximum value in thepresence of 0.1 M NaC1,25 The fact that theupper asymptote shiftstoward the left with
mM CaClz can be explained by a sharp increase of the K, increased concentration of cations while the lower one re-
value to itsmaximum combined with a more gradual increase mains almost unchanged (except at high cation concentra-
of Kl (Fig. 3). A similar effect has been observed in chloro- tion) indicates that cations bind to hemoglobin at late stages
cruorin (28). of oxygenation. These features are consistent the results
with
Similar shapes of the pH dependence of n have been
- obtained for A. marina hemoglobin (12) and P. leptochaeta
reported for several extracellular hemoglobins and chloro- chlorocruorin (28) and seem to be a general characteristic of
cruorins from different annelid species (8-12, 16-23, 37, 39- extracellular hemoglobins and chlorocruorins of annelids.
45) where the pHgiving a maximum n varies from pH 7.5 to Magnetic quadrupole relaxation experiments suggest that
9.0. Chiancone et al. (35) reported an exceptional case where Na+ may compete with Ca" for the same site in Lumbricus
the shape of pH dependence of the Hill coefficient is concave sp.' hemoglobin (45). The stoichiometry of Ca2+binding was
upward as observed for %z subunits from Affinis affiinis(44). estimated to be 0.26-0.31 Caz+/heme. These values are very
Effect of Salts-The present experiments show that the close to our values: 0.33 M$+ and 0.37 Ca2+ions taken up per
effect of different salts on oxygen binding are specific and oxygen bound (Table 11). Chiancone et al. (20) reported that
depend exclusively on the cations. Comparison of data at the 1.6 oxygenation-linked Ca2+ions/heme occur in the pH range
same c1- concentrations (Fig. 4B), one set at 50 m CaClz
M 7.7-8.5 and suggested that carboxyl groups with abnormal pK
and the other at mM NaCl or at the corresponding ionic
100 values may be responsible for the Ca2+-binding site for A.
strength of NaCl (150 mM), shows clearly that the increase ornuta hemoglobin. However, it seems rather unlikely that
in oxygen affinity iscaused by Ca2+,not by C1-. The data also carboxyl groups themselves would have pK values this high.
show that theeffects cannot be explained merely in terms of Makino (46) showed for hemocyanin from Dolabella auricu-
changes in ionic strength. The absence of an effect of C1- on laria that H+ and may compete for the same binding site
Ca2+
oxygenation issupported by nuclear magnetic quadrupole on the basis of measurements of equilibrium dialysis and H+
relaxation experiments, where C1- binds to both liganded and titration. He also suggested that theCaz+-bindingsite(s) may
unliganded forms of the hemoglobin with the same affinity include a histidine residue on the basis of the calculated pK
(45). The stronger effect of divalent than monovalent cations value of the protonated site.
is probably also true for other extracellular hemoglobins and Our data (lower panel in Fig. 2) suggest that themaximum
chlorocruorin (12, 20, 28). Among divalent cations, Ca2+has number of oxygenation-linked protons apparently depends
8. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8421
on ions such as Na+ or Ca2+ and reciprocally, the protons contains 70 Ca2+relative to 160 iron atoms. The sample was
which are released in the presence of Na+ or Ca" depend on dialyzedexhaustively against distilled deionizedwater or
pH. Ourobservations are consistent with those of Santucci et against 10 m EDTA, pH 7, followedby dialysis against
M
a. (15) on the hemoglobin of Octolasium cornplanaturn. We
l water. Chiancone et al. (45) suggested that Ca2+acts as a
suggest that both sets of data may be explained in follow-
the cross-linking agent between two carboxyl groups anchored at
ing way.We suggest that certain oxygenation-linkedacid the interface between two "one-twelfth"subunits. The whole
groups are present which have highpK values (at least 9.5 or molecule dissociates to %z subunits at alkaline pH, but this
higher ). If cations could bind only to thedeprotonated group dissociation is prevented by Ca2+ions (49). The whole mole-
with even a low affinity the fraction of molecules with the cules of some hemoglobins dissociate evenneutral pH upon
at
acid groups dissociated would be greatly increasedat a much removing Ca" ions (20, 50). Onecan, therefore, ask whether
lower pH than would otherwisebepossible. Assume the "structural" Ca2+ and "functional" Ca" have the same or
following simple sequence illustrative purposes,
for different binding sites. Chiancone et al. (20) suggested that
structural binding sites differ from functional ones on the
basis that a concentration of Ca2+ions only slightly above1
m Ca2+is enough to stabilize the whole structure whereas
M
where K, is the acid dissociation constant K z is the binding more than 10 mMCa" is necessary to effect a change in the
and
constant forthe cation. If the apparent dissociation constant in functional properties. It would, therefore, seem to be impor-
the presence of cations is given by Ki = ([Hb] + [HbNa]) [H+]/ tant to determine the level of subunit dissociation at which
[HbH+]then the apparent pK; will be given by functional alteration by Ca2' can be observed. If L. terrestris
pK; = pKl - log(1 + Kz[Na+]) hemoglobin wereto have the same number bound Ca" ions
of
as T. tubifex hemoglobin, 0.44/heme (48), it would mean that
If Na+ = 0.3 M and we assume only a very modest binding deoxygenation would be associated with the dissociation of
constant for Na+, say 100, this would be sufficient to lower most of the Ca" since 0.37 Ca2+ ions become boundper
the pKl value by 1.5 units. The greater effects of Ca" could oxygen bound or one Ca+ per 3 0 . this were true then at
, If
be explained by a much larger value of K2. Since the log P ~ o least 84% of the Ca2+would be functional and thepossibility
values at high and low pH in the presence of sufficient salt is raised that uniquely structural nonfunctional Ca2+may not
differ by about 1.1, the value of the product KlK2would shift exist. The lowest concentration of added Ca" which we have
by the same amount because of the linked-function relation- used is 0.2 mM. The estimated amount of endogenous bound
ship. One cannot tell on the basis of the present data whether ca2' is no more than 26 pM (0.44 X 60 pM) or 13% of that
values of Kl and K, are both oxygenation dependent. This added, an amount that appears to be too small to affect our
simple model is probably a considerable oversimplification Bohr proton calculations.
but suffices to show that cations can lower the pH at which Weber and Olsen (51) have recently sought to explain the
the Bohr effect couldbe observed. Analternative would be to dependence of oxygen binding on cations by invoking a con-
assume that the cation binds first and that this results in a cept of "surface pH." They argue that the cations do not exert
lowered pK for the acid groups. The net result would be the their effect by binding to specific sites but by altering the
same. surface pH. The basis of this conclusion rests on a Gouy-
Thedata indicate that approximately 0.77 protons are Chapman planar model with a fixed uniform charge density
released and 0.37 Ca2+are taken up per oxygen bound at pH on the surface. However, amino acid sequence and composi-
7.4 almost exactly 2 protons/calcium ion. Similarly, for mon- tion data indicate that the net negative charge results from
ovalent ions, 0.52 Na+ ions are taken up and 0.54 protons only a small difference between largenumbers of both posi-
released per0 2 bound givinga ratio of 1:l. Although the Bohr tively and negatively charged groups which havevery non-
a
effect in the presence of Li+ has not been measured, it is uniform distribution. This means that any electrostaticeffects
striking that the number of Li+ ions taken up per 0,bound on ion distribution must be extremely local. Although such
at pH 7.4 is 0.75, close to thevalue forthe Bohr effect in the effects would give rise to local Debye-Hiickeldistributions of
presence of calcium. These results indicate the presence of ions, the concept of surface pH does not appear physically
two acid groups.Since a Ca2+ ion may becoordinated with up meaningful. Furthermore, the very high mobility of protons
to 8 ligands (47)an attractivepossibility is that two salt links would tend to homogenize this effect. Weber and Olsen (51)
of the form, -NH,+ -0OC-, are present. The pK value of the excluded binding at specific sites largely on the basis that
assumed -NH,+ wouldbe raised to over 10 because of the choline and Na+ appeared to have the same effect even though
influence of the -COO- group. Ionization of two such groups the two cations differ greatlyin size. This could be fortuitous
might lead to a binding site for Ca2+ with least four groups. because our
at data on four different divalent cations (Ca2+,Sr2+,
Ba", M P )show that each has a unique effect which cannot
be explained the basis of ionic strength. Rather, the effects
on
appear to depend at least in parton ionic radius. Monovalent
ions (Li+, Na+, K+) also have specific effects; the number
-NH; i "OOC bound per O2 bound is closely related to ionic radius: the
smallest Li+ is bound to the greatest extent, Na+ is next,
The Ca2+ could coordinate with other protein groups such followed bythe largest ion, K'. Although the effects of cations
also
as the carbonyl group of the peptide bond or other amino acid on annelid hemoglobins must involve electrostatic binding,
side chains and/or with water molecules (47). This model this does not excludespecificity.Detailed studies of pH-
would explain nicely the observed relationship between the dependent processes in many proteins (52) indicate that a
Bohr effect and the binding of both monovalent and divalent primary role is played by electrostatic modifications of pK
cations. values and by conformation-dependentchanges in hydrogen
The assembly of extracellular hemoglobins fromtheir con- bonding.
stituent subunits is well known to depend on Ca2+. Rokosz Reversibility of Oxygenation Properties upon pH
and Viogradov (48) reported on the basis of x-ray fluores- Change-Giardina et al. (10) concluded that "native" hemo-
cence that thewhole molecule of Tubifex tubifex hemoglobin globin of the earthworm' was in a metastable conformation
9. 8422 Oxygenation Properties of Lumbricus terrestris Hemoglobin
which was converted irreversibly to a more stable state when ent enthalpy of oxygenation in Lumbricus hemoglobin will
the Hb solution was brought to neutral pH after incubation also include contributions from proton reactions. Since Bohr
at either pH 6.0 or 10 for 1 h. The basis of this conclusion proton release in Lumbricus hemoglobin is coupled to cation
was the finding that this treatment appeared to decrease the binding, the enthalpy of cation binding will also be involved.
value of the Hill coefficient irreversibly.Several workers (35- Functional Unit-Since the hemoglobin of L. terrestris has
37) have supported the idea of “a metastable state” by observ- about 200 02-binding subunits the task of describing the
ing such an irreversible decrease in n. This phenomenon was oxygen equilibrium directly in terms of a 200-step Adair model
also believed to be supported by circular dichroism (53) and is clearly hopeless. However,if it could be demonstrated that
small-angle neutron-scattering experiments (36).However, cooperative oxygen binding involved only small set of inter-
a
our results (Fig. 8 and Table 111) show that the reduction in acting groups, the problem would be easier to handle. Several
cooperativity upon exposure to acid or alkaline pH is com- attempts havebeenmade to estimate the size of such a
pletely reversible. The MetHb index shows that themanipu- functional unit in annelid hemoglobins and chlorocruorins.
lation involved in this experiment such as equilibration with Weber (16) reported that at least five 02-binding sites in A.
Sephadex G-25 or incubation in dilute solution of HbOz at marina hemoglobin constitute an interacting group. Wyman
25 “C increased the MetHb content compared to that of (29) reported at least 5 interacting sites for Spirographis
untreated Hb. In the case of incubation at pH 9:0 without spallanzanii chlorocruorin and Colosimo et al. (59) reported
Ca2+, n value decreasedsignificantly, apparently irrevers-
the 10 sites for the same chlorocruorin. I a and Yoshikawa (28)
mi
ibly. Such a drop in the MetHbindex is serious. Fig. 6 shows reported 6 interacting sites for P. leptochaeta chlorocruorin.
thatthe presence of MetHb reduces the value buthas All of these estimates have been based on the MWC model
little effect on the P, value. We conclude, therefore, that and/or deductions based onthe Hill coefficient. We have also
”irreversible” decrease in n at pH 9.0 without Ca2+, as
- attempted to estimate the size of the functional unit on these
observed in this experiment, was caused by the formation of bases. A plot of oxygen equilibrium data interms of a linear-
MetHb. Presumably the MetHb formation would have bees izedMWC equation (Equation 6) is shown in Fig.9. The
even higher at pH 10. Our results are consistent with the value of X in the figure is calculated from the relation, X =
report that Ca2+prevents dissociation of the whole molecule (1 + KTP)/(l + KRP).Although the data are nonlinear, the
and also protects against autoxidation (48,53-55). maximum slope in the central part was used to provide the
Martel et al. (36) reported an irreversible decrease in the values shown in Table I. These data show that the apparent
Hill coefficientfor L. terrestris hemoglobin that they ex- number of interacting sites appears to vary between 5 and 12,
plained as resulting from mismatched reassembly upon re- depending on pH. Maximum values were obtained between
turning the solution to neutral pH. Carbon monoxide was pH 7.8 and 8.2. This corresponds to the pH range which gave
apparently not used as a protective agent during hernoglobin the maximum valuesof n. Similar estimates of the number of
preparation and storage (56), and Ca2* appears not to have interacting sites were obtained with Kegeles’ method (32) and
beenusedwhenhemoglobinwasdialyzed against pH 9.0 are listed in Table I together with y,,. The datasuggest that
buffer (36).A significant amount of methemoglobin can form the size of the functional unit depends not only on the pH
under these conditions, as described above. We believe, there- but also on the kind and concentration of salt. Although the
fore, that theapparently irreversible decrease in the value of calculations give plausible numbers, all of which are very
n may well result from the formation of methemoglobin. small compared to the total number of hemes/molecule, they
Circular dichroism experiments (53) showed spectral are based on a two-state MWCmodel.Wehaveshown,
changes in the Soret region that were attributed to a local however, that a two-state model isnot sufficient for the
alteration of the heme environment. This local change, how- description of the data. The nonlinearity of the curves inFig.
ever, does not appear to exclude the possibility of MetHb 9 emphasizes this point. An alternative procedure would be
formation for the following reasons. Harrington et al. (57) to adopt a completely statistical approach to binding. More
reported for L. terrestris hemoglobin that the CD spectrum generally, without recourse to any model of hemoglobin be-
has maxima at 412 and 422 nm in theoxy and cyanmet forms, havior, nmax be shown to be proportional to thestatistical
can
respectively. Ascoli al. (25) reported that theaquomethem-
et
oglobin form at pH 7.0 irreversibly changed to a hemichrome
after bringing the pH to 6.0 or 7.6. This hemichrome may
cause a change in the heme environment which would result
in a different CD spectrum. Although small-angle neutron-
scattering data (36) show a scattering curve different from
that of the parent molecule, the difference is difficult to
interpret unambiguously.
-
In contrast to the irreversible changes reported for “Lum-
bricus SP.’’~ (IO), Santucci et al. (15) and Chiancone et al.
Hb
(20) have reported reversible changes in n with pH for the
hemoglobins of both 0. complanatum and A. ornata. These
findings together with our data suggest that the existence of
a metastable state of ferrous hemoglobin is unlikely.
Temperature Effect-The variation of A H S O with pH is very
similar tothat of human HbA (Table IV). An enthalpy
-2t i
difference of 3.5 kcal/mol observed at pH 9.0 between L. -2 -I 0
terrestris hemoglobin and human HbA is larger than those at log x
pH 6.6 (1.9 kcal/mol) and pH7.4 (1.8kcal/mol). Studies have FIG. 9. Plots of oxygen binding by. terrestris hemoglobin
L
shown (27, 58) that the intrinsic heat of heme oxygenation according to the linearized MWC equation(Equation 6 in
for human hemoglobin is -14.4 to -15.6 kcal/mol of oxygen text). Kl and K,,, (Table I) were used for KT and KB, respectively.
bound. Variation in the apparent enthalpy appears to result Buffer: 0.05 M BisTris/propane/HCl, 0 1 M NaC1.pH from left to
.
entirely from proton and other nonheme ligands. The appar- right: 8.10,7.35,8.86, 6.55.
10. Oxygenation Properties of Lumbricus terrestris Hemoglobin 8423
variance of the distribution of bound ligand among sites (60, 26. Antonini, E., and Brunori, M. (1971) i Hemoglobin and Myoglo-
n
61), and so a high Hill coefficient reflects a highly nonrandom bin in the Reactions with Ligands, North-Holland Publishing
distribution of bound ligands among possible sites. At least Co., Amsterdam
27. Imai, K. (1979) J. Mol. Biol. 133,233-247
four intrinsic 02-bindingequilibria must be involved because 28. Imai, K., and Yoshikawa, S. (1985) Eur. J. Biochem. 147, 453-
4 major kinds of subunits with unique 02-bindingcharacter- 463
istics have been isolated (1).One can hope to establish a 29. Wyman, J. (1964) Ado. Protein Chem. 19, 223-286
better basis upon which to describe the oxygen equilibria by 30. Monod, J., Wyman, J., and Changuex, J. P. (1965) J. Mol. Biol.
obtaining detailed data on the subunits'in various states of 12,88-118
assembly and so to obtain a description of the whole molecule 31. Decker, I., Sabel, A., Linzen, B., and Van Holde, K. E. (1983) in
Life Chemistry Reports (Wood, E. J., ed) Suppl. 1,pp. 251-256,
in terms of its 4 major constituent subunits and theirmeas- Harwood AcademicPublishers, London
urable interactions. This procedure will also help ascertain 32. Kegeles, G. (1979) FEBS Lett. 103,5-6
whether the minor chains V and VI (Ref. 6) play any impor- 33. Imai, K. (1982) Allosteric Effects in Haemoglobin, pp. 113-114,
tant functional or structural role. Cambridge University Press, London
34. Imai, K., Yonetani, T., and Ikeda-Saito, M. (1977) J Mol. Biol.
'
?
Acknowledgments-We wish to express our thanks to I. Tyuma
Dr. 109,83-97
of the Medical School at Osaka University and Dr. H. Morimoto, 35. Chiancone, E.,Ascoli,F., Giardina, B., Vecchini, P., Antonini,
Faculty of Engineering Science, Osaka University for their interest E., Musmeci, M. T., Cinb, R., Zagra, M., D'Amelio, V., and De
and encouragement. We thank Dr. Walter J. Harman (Dept. of Leo, G. (1977) Biochim. Biophys. Acta 4 9 4 , 1-8
Zoology, Louisiana State University) for the identification of the 36. Martel, P., Powell, B. M., Kapp, 0. H., and Vinogradov, S. N.
worms. (1982) Biochim. Biophys. Acta 709,134-141
37. Frossard, P. (1982) Biochim. Biophys. Acta 704,524-534
38. Imai, K., and Yonetani, T (1975) J. Biol. Chem. 250,7093-7098
.
REFERENCES 39. Wood, E. J., Mosby, L. J., and Robinson, M. S. (1976) Biochem.
1. Fushitani, K., Imai, K., and Riggs, A. F. (1985) in Symposium on J. 153,589-596
Invertebrate Oxygen Carriers (Linzen, B., ed) Springer Verlag, 40. Bannister, J. V., Bannister, W. H., Anastasi, A., and Wood, E. J.
New York, in press (1976) Biochem. J. 159,35-42
2. Imai, K., Yoshikawa, S., Fushitani, K., Takizawa, H., Handa, T., 41. Garlick, R. C., and Terwilliger, R. C. (1977) Comp. Biochem.
and Kihara, H. (1985) in Symposium on Invertebrate Oxygen Physiol. 57B, 177-184
Carriers (Linzen, B., ed) Springer Verlag, New York, in press 42. Weber, R. E., Bonaventura, J., Sullivan, B., and Bonaventura, C.
3. Chung, M. C. M., and Ellerton, H. D. (1979) Prog. Biophys. Mol. (1978) J. Comp. Physiol. 123,177-184
Biol. 35,53-102 43. Warren, L. M., Wells, R. M. G., and Weber, R. E. (1981) J. Exp.
4. Kapp, 0. Vinogradov, S. N., Ohtsuki, M., and Crewe, A.V.
H., Mar. Bwl. Ecol. 5 5 , 11-24
(1982) Biochim. Biophys. Acta 704,546-548 44. Chung, M. C. M., and Ellerton, H. D. (1982) Biochim. Bwphys.
5. Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257,9005- Acta 702,17-22
9015 45. Chiancone, E., Bull, T. E., Norne, J. E., Forsen, S., and Antonini,
6. Kapp, 0. Polidor, G., Mainwaring, M. G., Crewe, A.V., and
H., E. (1976) J. Mol. Biol. 107,25-34
Vinogradov, S. N. (1984) J. Biol. Chem. 259,628-639 46. Makino, N. (1972) J. Biochem. (Tokyo) 71,987-991
7. Haughton, T. M., Kerkut, G.A., and Munday, K. A. (1958) J. 47. Williams, R. J. P. (1976) Symp. Soc. Exp. Bwl. 3 0 , 1-17
EXP.BWl, 35,360-368 48. Rokosz, M. J., and Vinogradov, S. N. (1982) Bwchim. Biophys.
8. Manwell, C. (1959) J. Cell. Comp. Physiol. 53, 61-74 Acta 707,291-293
9. Cosgrove,W.B., and Schwartz, J. B. (1965) Physiol. 2001. 38, 49. David, M. M., and Daniel, E. (1974) J. Mol. Biol. 8 7 , 89-101
206-212 50. Terwilliger, R. C., Terwilliger, N. B., and Roxby, R. (1975) Comp.
10. Giardina, B., Chiancone, E., and Antonini, E. (1975) J. Mol. Biol. Bwchem. Physiol. 50B,225-232
93,l-10 51. Weber, R. E., and Olsen, L.F. (1984) Mol. Physiol. 6 , 1-8
11. Vinogradov, S. N., Shlom, J. M., Hall, B.C., Kapp, 0. and H., 52. Matthew, J. B., Gurd, F. R. N., Garcia-Moreno, E., Flanagan, M.
Mizukami, H. (1977) Biochim. Biophys. Acta 4 9 2 , 1 3 6 1 5 5 S.
A., March, K. L., and Shire, J. (1985) CRC Crit. Rev. Bwchem.
12. Weber, R. E. (1981) Nature 292,386-387 18391-197
13. Imai, K. and Yonetani, T. (1975) J. Biol. Chem. 250,2227-2231 53. Ascoli, F., Chiancone, E., and Antonini, E. (1976) J. Mol. Biol.
14. Imaizumi, K., Imai, K., and Tyuma, I (1979) J. Biochem. (Tokyo)
. 105,343-351
86,1829-1840 54. Swaney, J. B., and Klotz, I. M. (1971) Arch. Bwchem. Biophys.
15. Santucci, R., Chiancone, E., and Giardina, B. (1984) J. Mol. Biol. 147,475-486
179,713-727 55. Chiancone, E., Brenowitz, M., Ascoli, F., Bonaventura, C., and
16. Weber, R. E. (1970) Comp. Biochem. Physiol. 35,179-189 Bonaventura, J. (1980) Biochim. Biophys. Acta 623,146-162
17. Terwilliger, R. C. (1974) C o w . Bwchem. Physiol. 48A, 745-755 56. Shlom, J. M., and Vinogradov, S. N. (1973) J. Biol. Chem. 2 4 8 ,
18. Garlick, R. L., and Terwilliger, R.C. (1975) Comp. Biochem. 7904-7912
Physiol. 5 1A, 849-857 57. Harrington, J. P., Pandolfelli, E. R., and Herskovits, T. T. (1973)
19. Weber, R. E. (1975) J. Comp. Physwl. 9 9 , 297-307 Biochim. Bwphys. Acta 3 2 8 , 61-73
20. Chiancone, E., Femzzi, G., Bonaventura, C., and Bonaventura, 58. Mills, F. C., Ackers, G. K., Gaud, H. T., and Gill, S. J. (1979) J.
J. (1981) Biochim. Biophys. Acta 670,84-92 Biol. Chem 254,2875-2880
21. Chung, M. C. M., and Ellerton, H. D. (1982) Biochim. Biophys. 59. Colosimo, A., Brunori, M., and Wyman, J. (1974) Biophys. Chem.
Acta 702,6-16 2,338-344
22. Ochiai, T. (1983) Arch. Biochem. Biophys. 226, 111-117 60. Cohn. E. J.. and Edsall. J. T. (1943) Proteins. Amino Acids and
23. Ochiai, T. (1984) Arch. Biochem. Biophys. 2 3 1 , 136-143 Peptides & Ions and Dipolarons,'pp. 462-463, Reinhold Pub-
24. Imai. K. (1981) Methods Enzvmol. 76.438-449
. . lishing Corp., New York
25. Ascoli, F., Rossi-Fanelli, M. R., Chiancone, E., Vecchini, P., and 61. Edsall, J. T., and Gutfreund, H. (1983) Biothermodynumics, p.
Antonini, E. (1978) J. Mol. Biol. 119, 191-202 201, John Wiley and Sons, New York