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.
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 summarizes the total synthesis of 2,6-dideoxy-2,6-imino-7-O-β-D-glucopyranosyl-D-glycero-L-gulo-heptitol hydrochloride (8), a potent inhibitor of α-glucosidases. The key steps involve homologation and amination of 2,3,4,6-tetra-O-benzyl-D-glucopyranose (1) to form the protected amine 4. An intramolecular cyclization of 4 catalyzed by mercuric acetate formed the piperidine ring. Glycosylation of aglycon 6 with acetob
There are five types of skeletal rearrangements: electron deficient, electron rich, radical, rearrangements on aromatic rings, and sigmatropic rearrangements. Molecular rearrangements involve the migration of a group from one atom to another within the same molecule. Examples include the Wagner-Meerwein rearrangement, pinacol-pinacolone rearrangement, and Cope rearrangement. Rearrangements are driven by stability of the carbocation intermediate or relief of ring strain.
This study investigated the polymerization of lactic acid as a model for prebiotic peptide formation via ester-amide exchange. Lactic acid was polymerized in a closed system at 85°C over various time points. HPLC and 1H-NMR were used to analyze the polymers and determine degree of polymerization (DP) and total lactic acid units. DP was found to increase with time while total units decreased, showing polymer regeneration. Methods showed consistent results within 10-15% error. Further studies will compare kinetics to a computer simulation to determine rate constants and model polymerization from various monomers.
Carbon−Heteroatom Coupling Using Pd-PEPPSI Complexes (1)DrMAdamSayah
This document summarizes recent advances in using Pd-PEPPSI complexes as catalysts for aryl amination and aryl sulfination reactions. Pd-PEPPSI-IPent was found to be an effective catalyst for aryl sulfinations, enabling the reactions to proceed at 40°C. Further modifications to the pyridine and NHC ligands lowered the activation temperature of the precatalyst to as low as 40°C, allowing the aryl sulfinations to be carried out under very mild conditions. The improved steric environment around Pd is believed to facilitate the initial reductive elimination step needed to generate active Pd(0) and enter the catalytic cycle.
The document discusses various types of molecular rearrangement reactions. It begins by defining rearrangement reactions as those where the atoms or groups in a molecule reshuffle to form a structural isomer of the original substance. Rearrangements are then classified as intermolecular or intramolecular. Several examples of nucleophilic rearrangements are provided, including carbonium ion rearrangements like the pinacol-pinacolone, Wagner-Meerwein, and benzillic acid rearrangements. Nitrogen deficiency rearrangements like the Schmidt, Curtius, Hoffmann, Beckmann, and Lossen rearrangements are also briefly described. The mechanisms and features of several important rearrangements are discussed in more detail.
This document discusses alkenes, including:
1. Alkenes have one or more double bonds between carbon atoms and are named using IUPAC rules based on carbon chain length and double bond position.
2. Physical properties of alkenes include higher boiling points with increased carbon chain length and lower boiling points for branched isomers due to decreased intermolecular forces.
3. Alkenes undergo electrophilic addition reactions due to the electron density of the carbon-carbon double bond, allowing attack by electrophiles to form carbocations and adding nucleophiles in a stepwise mechanism.
Free radicals are atoms or groups of atoms with unpaired electrons that can participate in chemical reactions. Common examples include hydroxyl (HO·) and methyl (H3C·) radicals. Free radical reactions typically occur by initiation, propagation, and termination steps in a chain reaction mechanism. The stability of free radicals is determined by factors such as conjugation, hyperconjugation, and hybridization. Radicals can be detected using techniques like electron paramagnetic resonance spectroscopy.
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 summarizes the total synthesis of 2,6-dideoxy-2,6-imino-7-O-β-D-glucopyranosyl-D-glycero-L-gulo-heptitol hydrochloride (8), a potent inhibitor of α-glucosidases. The key steps involve homologation and amination of 2,3,4,6-tetra-O-benzyl-D-glucopyranose (1) to form the protected amine 4. An intramolecular cyclization of 4 catalyzed by mercuric acetate formed the piperidine ring. Glycosylation of aglycon 6 with acetob
There are five types of skeletal rearrangements: electron deficient, electron rich, radical, rearrangements on aromatic rings, and sigmatropic rearrangements. Molecular rearrangements involve the migration of a group from one atom to another within the same molecule. Examples include the Wagner-Meerwein rearrangement, pinacol-pinacolone rearrangement, and Cope rearrangement. Rearrangements are driven by stability of the carbocation intermediate or relief of ring strain.
This study investigated the polymerization of lactic acid as a model for prebiotic peptide formation via ester-amide exchange. Lactic acid was polymerized in a closed system at 85°C over various time points. HPLC and 1H-NMR were used to analyze the polymers and determine degree of polymerization (DP) and total lactic acid units. DP was found to increase with time while total units decreased, showing polymer regeneration. Methods showed consistent results within 10-15% error. Further studies will compare kinetics to a computer simulation to determine rate constants and model polymerization from various monomers.
Carbon−Heteroatom Coupling Using Pd-PEPPSI Complexes (1)DrMAdamSayah
This document summarizes recent advances in using Pd-PEPPSI complexes as catalysts for aryl amination and aryl sulfination reactions. Pd-PEPPSI-IPent was found to be an effective catalyst for aryl sulfinations, enabling the reactions to proceed at 40°C. Further modifications to the pyridine and NHC ligands lowered the activation temperature of the precatalyst to as low as 40°C, allowing the aryl sulfinations to be carried out under very mild conditions. The improved steric environment around Pd is believed to facilitate the initial reductive elimination step needed to generate active Pd(0) and enter the catalytic cycle.
The document discusses various types of molecular rearrangement reactions. It begins by defining rearrangement reactions as those where the atoms or groups in a molecule reshuffle to form a structural isomer of the original substance. Rearrangements are then classified as intermolecular or intramolecular. Several examples of nucleophilic rearrangements are provided, including carbonium ion rearrangements like the pinacol-pinacolone, Wagner-Meerwein, and benzillic acid rearrangements. Nitrogen deficiency rearrangements like the Schmidt, Curtius, Hoffmann, Beckmann, and Lossen rearrangements are also briefly described. The mechanisms and features of several important rearrangements are discussed in more detail.
This document discusses alkenes, including:
1. Alkenes have one or more double bonds between carbon atoms and are named using IUPAC rules based on carbon chain length and double bond position.
2. Physical properties of alkenes include higher boiling points with increased carbon chain length and lower boiling points for branched isomers due to decreased intermolecular forces.
3. Alkenes undergo electrophilic addition reactions due to the electron density of the carbon-carbon double bond, allowing attack by electrophiles to form carbocations and adding nucleophiles in a stepwise mechanism.
Free radicals are atoms or groups of atoms with unpaired electrons that can participate in chemical reactions. Common examples include hydroxyl (HO·) and methyl (H3C·) radicals. Free radical reactions typically occur by initiation, propagation, and termination steps in a chain reaction mechanism. The stability of free radicals is determined by factors such as conjugation, hyperconjugation, and hybridization. Radicals can be detected using techniques like electron paramagnetic resonance spectroscopy.
The document summarizes various organic reaction mechanisms including:
1) Free radical substitution, electrophilic addition, nucleophilic substitution, elimination, addition-elimination, electrophilic substitution, esterification, alkaline hydrolysis, nucleophilic addition.
2) Specific mechanisms are described for hydration of alkenes, addition polymerization, bromination of alkenes, nucleophilic substitution, elimination, dehydration, esterification.
3) The formation of polymers like polyamides, polyesters through reactions of dibasic acids and diamines or diols are summarized.
Proton euilibria in minor groove of dnamganguly123
1) The document describes an experiment testing the prediction that regions of increased hydrogen ion density exist in the grooves of DNA. Probes with variable linker lengths and a proton-sensitive carboxyl group were attached to DNA in the minor groove.
2) The apparent pKa values of the carboxyl groups were higher than in free solution, increasing with shorter linker lengths. This agrees with calculations showing higher hydrogen ion density in the grooves.
3) The experiment provides experimental evidence supporting the theoretical prediction of acidic domains with elevated hydrogen ion density in the DNA minor groove.
This document discusses pKa values, which refer to the acid dissociation constant of acids. It defines pKa and explains how pKa values can be used to understand and predict the behavior of acids and bases in solution. The document covers several key topics in pKa including the factors that influence pKa values, common types of acids like mono- and polyprotic acids, and the significance and applications of pKa in fields like chemistry, biology, and medicine.
This document summarizes a study on the kinetics and mechanism of the acetyl transfer partial reaction of the acetyl-CoA decarbonylase/synthase (ACDS) complex from Methanosarcina barkeri. The study found that acetyl transfer activity required strongly reducing conditions and exhibited a ping-pong kinetic mechanism consistent with formation of an acetyl-enzyme intermediate. Analysis of inhibitory effects provided values for substrate binding and isotope exchange experiments provided information about the stability of the acetyl-enzyme intermediate. The results help characterize the acetyl-enzyme intermediate and have implications for understanding the mechanism of C-C bond cleavage catalyzed by the ACDS complex.
The document discusses several rearrangement reactions including the pinacol rearrangement, Beckmann rearrangement, Heck reaction, ozonolysis, and Grignard reaction. The pinacol rearrangement involves the acid-catalyzed rearrangement of vicinal diols to ketones or aldehydes. The Beckmann rearrangement converts ketoximes to N-substituted amides. The Heck reaction is a palladium-catalyzed coupling of aryl or alkenyl halides with alkenes. Ozonolysis uses ozone to cleave alkenes and alkynes, replacing the multiple bond with a carbonyl. Grignard reagents are important in organic synthesis.
The haloalkanes are a group of chemical compounds derived from alkanes containing one or more halogens. They are a subset of the general class of halocarbons, although the distinction is not often made. Haloalkanes are widely used commercially and, consequently, are known under many chemical and commercial names.
This document discusses electrophilic aromatic substitution and nucleophilic aromatic substitution reactions. It covers topics such as the directing effects of substituents in electrophilic aromatic substitution, including ortho/para directing activating groups and meta directing deactivating groups. Reaction mechanisms are shown for common electrophilic aromatic substitutions like bromination, nitration, sulfonation, and Friedel-Crafts acylation and alkylation. Side chain reactions and the limitations of Friedel-Crafts reactions are also discussed. Finally, the document addresses nucleophilic aromatic substitution reactions and the criteria for "SNA" reactivity, including the benzyne intermediate that can be involved.
The overall rate equation for this reaction is:
Rate = k[R-R-OH][H2O]
Where k is the rate constant and [R-R-OH] and [H2O] are the concentrations of the reactants R-R-OH and H2O, respectively.
This document discusses the reactivity of various carboxylic acid derivatives including alkanoyl halides, anhydrides, esters, amides, and alkanenitriles. It compares their relative reactivities and describes common reaction mechanisms. Alkanoyl halides are the most reactive and undergo nucleophilic substitution reactions. Anhydrides and esters undergo similar reactions but are less reactive. Amides have lower acidity than esters due to resonance and undergo hydrolysis or reduction. Alkanenitriles undergo hydrolysis to carboxylic acids or reactions with organometallic reagents to form ketones or aldehydes.
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 discusses organosulfur compounds, including their preparation, reactions, and uses. Key points:
- Organosulfur compounds contain sulfur bonded to carbon atoms. Common examples include cysteine and methionine amino acids.
- Thiols and thioethers are important organosulfur compounds that can be prepared from alkyl halides, alcohols, or sodium salts of thiols.
- Thioethers undergo substitution and oxidation reactions. Dimethyl sulfide can be oxidized to dimethyl sulfoxide and further to dimethyl sulfone.
- Sulfur ylides derived from sulfonium and sulfoxonium salts react with carbon
This is Power Point Presentation on Topic "Electrophilic Aromatic Substitution Reactions" as per syllabus of "University of Mumbai" for S.Y. B. Pharmacy (Sem.: IV) students.
This document summarizes information about alcohols, alkyl halides, ethers, and epoxides. It describes their structures, naming conventions, physical properties, industrial sources, and common laboratory preparation methods. For alcohols, it discusses primary, secondary and tertiary classifications. Common reactions include cleavage of the C-OH and O-H bonds. Alkyl halides are described as R-X compounds that are insoluble in water. Ethers have the general formula R-OR' and are named by identifying the two groups attached to oxygen. Epoxides contain a three-membered ring structure.
1) The document discusses various organic chemistry reaction mechanisms including SN1, SN2, addition and substitution reactions.
2) It provides step-by-step explanations of the mechanisms of reactions such as hydrolysis of ethers with HI, addition of Grignard reagents to carbonyls, and acid-catalyzed dehydration of alcohols.
3) Limiting factors for different reaction mechanisms are discussed, such as steric hindrance preventing SN2 in tertiary halides and stability of carbocation intermediates determining SN1 rates.
Presentation is the basic summary of the research paper on the topic of ''Opposing effects of Na+ and K+ on the thermal stability on Na+ K+ ATPase activity''
This document presents a reaction mechanism for the atmospheric photochemical oxidation of benzene initiated by reaction with hydroxyl radicals. It develops an elementary reaction mechanism including 29 reactions and 26 species. Rate constants and thermodynamic parameters are analyzed using quantum Rice-Ramsperger-Kassel theory and group additivity techniques to determine equilibrium concentrations of reaction intermediates and product formation rates under atmospheric conditions. The mechanism accounts for important reaction intermediates like benzene-OH adducts and their reactions leading to ring-opening products such as dicarbonyl compounds.
Alkynes are hydrocarbons with a triple bond between two carbon atoms. Common alkynes include acetylene (C2H2), propyne, butyne, pentyne, etc. Their molecular formulas follow the pattern of CnH2n-2. Alkynes are named based on the number of carbons and whether the chain is straight or branched. They are generally reactive due to the triple bond. Alkynes undergo addition, polymerization, substitution, and combustion reactions. They can also form isomers based on chain structure or carbon position.
This document summarizes research on using commercial chiral anion-exchange LC columns packed with quinidine or quinine ligands to separate enantiomers of acidic drugs and related compounds using hydro-organic mobile phases. Key findings include:
1) Low pH mobile phases provided the best retention and enantioresolution. Selectivity was largely independent of mobile phase variables except at pH >5-6.
2) Enantioseparation was achieved for a range of drug acids including NSAIDs and mandelic acids. However, enantioselectivity was not sufficient to explore achiral-chiral separations in a single column.
3) Retention was very similar across the three columns tested,
This document describes the discovery of the first organocatalytic α,β,γ-trioxygenation of enals. The reaction proceeds through an initial TEMPO-mediated γ-oxygenation, followed by rapid racemization and reversible conjugate addition of water. This sets the stage for a second TEMPO incorporation at the α-position to set all three stereocenters. Using a tryptophan-derived imidazolidinone catalyst in fluorinated aromatic solvents, α,β,γ-trioxyaldehydes were obtained in up to 51% isolated yield and 85:15 er. Substitution at the δ-position was tolerated, but not at the α, β,
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+.
Carboxy-terminal Degradation of Peptides using Perfluoroacyl Anhydrides : C-T...Keiji Takamoto
This document describes a new method for determining the carboxy-terminal (C-terminal) amino acid sequence of peptides using perfluoroacyl anhydride vapor. Exposure of peptides to the vapor at -20°C for 0.5-1 hours sequentially degrades the peptide from the C-terminus. Analysis of the truncated peptide fragments by fast-atom-bombardment mass spectrometry allows determination of the C-terminal sequence based on mass differences. The method provides C-terminal sequence information as a complement to the common Edman degradation method for amino-terminal sequencing. The perfluoroacyl anhydride vapor method results in more extensive C-terminal degradation than a previous method using perfluoric acid
The document summarizes various organic reaction mechanisms including:
1) Free radical substitution, electrophilic addition, nucleophilic substitution, elimination, addition-elimination, electrophilic substitution, esterification, alkaline hydrolysis, nucleophilic addition.
2) Specific mechanisms are described for hydration of alkenes, addition polymerization, bromination of alkenes, nucleophilic substitution, elimination, dehydration, esterification.
3) The formation of polymers like polyamides, polyesters through reactions of dibasic acids and diamines or diols are summarized.
Proton euilibria in minor groove of dnamganguly123
1) The document describes an experiment testing the prediction that regions of increased hydrogen ion density exist in the grooves of DNA. Probes with variable linker lengths and a proton-sensitive carboxyl group were attached to DNA in the minor groove.
2) The apparent pKa values of the carboxyl groups were higher than in free solution, increasing with shorter linker lengths. This agrees with calculations showing higher hydrogen ion density in the grooves.
3) The experiment provides experimental evidence supporting the theoretical prediction of acidic domains with elevated hydrogen ion density in the DNA minor groove.
This document discusses pKa values, which refer to the acid dissociation constant of acids. It defines pKa and explains how pKa values can be used to understand and predict the behavior of acids and bases in solution. The document covers several key topics in pKa including the factors that influence pKa values, common types of acids like mono- and polyprotic acids, and the significance and applications of pKa in fields like chemistry, biology, and medicine.
This document summarizes a study on the kinetics and mechanism of the acetyl transfer partial reaction of the acetyl-CoA decarbonylase/synthase (ACDS) complex from Methanosarcina barkeri. The study found that acetyl transfer activity required strongly reducing conditions and exhibited a ping-pong kinetic mechanism consistent with formation of an acetyl-enzyme intermediate. Analysis of inhibitory effects provided values for substrate binding and isotope exchange experiments provided information about the stability of the acetyl-enzyme intermediate. The results help characterize the acetyl-enzyme intermediate and have implications for understanding the mechanism of C-C bond cleavage catalyzed by the ACDS complex.
The document discusses several rearrangement reactions including the pinacol rearrangement, Beckmann rearrangement, Heck reaction, ozonolysis, and Grignard reaction. The pinacol rearrangement involves the acid-catalyzed rearrangement of vicinal diols to ketones or aldehydes. The Beckmann rearrangement converts ketoximes to N-substituted amides. The Heck reaction is a palladium-catalyzed coupling of aryl or alkenyl halides with alkenes. Ozonolysis uses ozone to cleave alkenes and alkynes, replacing the multiple bond with a carbonyl. Grignard reagents are important in organic synthesis.
The haloalkanes are a group of chemical compounds derived from alkanes containing one or more halogens. They are a subset of the general class of halocarbons, although the distinction is not often made. Haloalkanes are widely used commercially and, consequently, are known under many chemical and commercial names.
This document discusses electrophilic aromatic substitution and nucleophilic aromatic substitution reactions. It covers topics such as the directing effects of substituents in electrophilic aromatic substitution, including ortho/para directing activating groups and meta directing deactivating groups. Reaction mechanisms are shown for common electrophilic aromatic substitutions like bromination, nitration, sulfonation, and Friedel-Crafts acylation and alkylation. Side chain reactions and the limitations of Friedel-Crafts reactions are also discussed. Finally, the document addresses nucleophilic aromatic substitution reactions and the criteria for "SNA" reactivity, including the benzyne intermediate that can be involved.
The overall rate equation for this reaction is:
Rate = k[R-R-OH][H2O]
Where k is the rate constant and [R-R-OH] and [H2O] are the concentrations of the reactants R-R-OH and H2O, respectively.
This document discusses the reactivity of various carboxylic acid derivatives including alkanoyl halides, anhydrides, esters, amides, and alkanenitriles. It compares their relative reactivities and describes common reaction mechanisms. Alkanoyl halides are the most reactive and undergo nucleophilic substitution reactions. Anhydrides and esters undergo similar reactions but are less reactive. Amides have lower acidity than esters due to resonance and undergo hydrolysis or reduction. Alkanenitriles undergo hydrolysis to carboxylic acids or reactions with organometallic reagents to form ketones or aldehydes.
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 discusses organosulfur compounds, including their preparation, reactions, and uses. Key points:
- Organosulfur compounds contain sulfur bonded to carbon atoms. Common examples include cysteine and methionine amino acids.
- Thiols and thioethers are important organosulfur compounds that can be prepared from alkyl halides, alcohols, or sodium salts of thiols.
- Thioethers undergo substitution and oxidation reactions. Dimethyl sulfide can be oxidized to dimethyl sulfoxide and further to dimethyl sulfone.
- Sulfur ylides derived from sulfonium and sulfoxonium salts react with carbon
This is Power Point Presentation on Topic "Electrophilic Aromatic Substitution Reactions" as per syllabus of "University of Mumbai" for S.Y. B. Pharmacy (Sem.: IV) students.
This document summarizes information about alcohols, alkyl halides, ethers, and epoxides. It describes their structures, naming conventions, physical properties, industrial sources, and common laboratory preparation methods. For alcohols, it discusses primary, secondary and tertiary classifications. Common reactions include cleavage of the C-OH and O-H bonds. Alkyl halides are described as R-X compounds that are insoluble in water. Ethers have the general formula R-OR' and are named by identifying the two groups attached to oxygen. Epoxides contain a three-membered ring structure.
1) The document discusses various organic chemistry reaction mechanisms including SN1, SN2, addition and substitution reactions.
2) It provides step-by-step explanations of the mechanisms of reactions such as hydrolysis of ethers with HI, addition of Grignard reagents to carbonyls, and acid-catalyzed dehydration of alcohols.
3) Limiting factors for different reaction mechanisms are discussed, such as steric hindrance preventing SN2 in tertiary halides and stability of carbocation intermediates determining SN1 rates.
Presentation is the basic summary of the research paper on the topic of ''Opposing effects of Na+ and K+ on the thermal stability on Na+ K+ ATPase activity''
This document presents a reaction mechanism for the atmospheric photochemical oxidation of benzene initiated by reaction with hydroxyl radicals. It develops an elementary reaction mechanism including 29 reactions and 26 species. Rate constants and thermodynamic parameters are analyzed using quantum Rice-Ramsperger-Kassel theory and group additivity techniques to determine equilibrium concentrations of reaction intermediates and product formation rates under atmospheric conditions. The mechanism accounts for important reaction intermediates like benzene-OH adducts and their reactions leading to ring-opening products such as dicarbonyl compounds.
Alkynes are hydrocarbons with a triple bond between two carbon atoms. Common alkynes include acetylene (C2H2), propyne, butyne, pentyne, etc. Their molecular formulas follow the pattern of CnH2n-2. Alkynes are named based on the number of carbons and whether the chain is straight or branched. They are generally reactive due to the triple bond. Alkynes undergo addition, polymerization, substitution, and combustion reactions. They can also form isomers based on chain structure or carbon position.
This document summarizes research on using commercial chiral anion-exchange LC columns packed with quinidine or quinine ligands to separate enantiomers of acidic drugs and related compounds using hydro-organic mobile phases. Key findings include:
1) Low pH mobile phases provided the best retention and enantioresolution. Selectivity was largely independent of mobile phase variables except at pH >5-6.
2) Enantioseparation was achieved for a range of drug acids including NSAIDs and mandelic acids. However, enantioselectivity was not sufficient to explore achiral-chiral separations in a single column.
3) Retention was very similar across the three columns tested,
This document describes the discovery of the first organocatalytic α,β,γ-trioxygenation of enals. The reaction proceeds through an initial TEMPO-mediated γ-oxygenation, followed by rapid racemization and reversible conjugate addition of water. This sets the stage for a second TEMPO incorporation at the α-position to set all three stereocenters. Using a tryptophan-derived imidazolidinone catalyst in fluorinated aromatic solvents, α,β,γ-trioxyaldehydes were obtained in up to 51% isolated yield and 85:15 er. Substitution at the δ-position was tolerated, but not at the α, β,
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+.
Carboxy-terminal Degradation of Peptides using Perfluoroacyl Anhydrides : C-T...Keiji Takamoto
This document describes a new method for determining the carboxy-terminal (C-terminal) amino acid sequence of peptides using perfluoroacyl anhydride vapor. Exposure of peptides to the vapor at -20°C for 0.5-1 hours sequentially degrades the peptide from the C-terminus. Analysis of the truncated peptide fragments by fast-atom-bombardment mass spectrometry allows determination of the C-terminal sequence based on mass differences. The method provides C-terminal sequence information as a complement to the common Edman degradation method for amino-terminal sequencing. The perfluoroacyl anhydride vapor method results in more extensive C-terminal degradation than a previous method using perfluoric acid
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.
This document summarizes an experiment to engineer ascorbate peroxidase (APX) activity into cytochrome c peroxidase (CCP) by introducing the APX ascorbate-binding site into CCP. Specifically, the researchers replaced the ascorbate-binding loop and a critical arginine residue in CCP with the corresponding residues from APX to create a mutant called CCP2APX. While wild-type CCP showed no APX activity, CCP2APX was able to catalyze the peroxidation of ascorbate, demonstrating that the engineered ascorbate-binding site could bind ascorbate. Crystal structures of CCP2APX confirmed that the engineered binding site
Bond-Specific Chemical Cleavages of Peptides & Proteins with Perfluoric Acid ...Keiji Takamoto
This document describes research on specific chemical cleavages of peptides and proteins using perfluoric acid vapors. The researchers established conditions for novel cleavages of certain peptide bonds, including glycyl-threonine, the amino side of serine residues, and the carboxyl side of aspartic acid residues. Exposure to vapors of various concentrations of heptafluorobutyric acid at different temperatures (30-90°C) resulted in selective cleavage of these bonds. The cleavages were studied using synthetic peptides as well as proteins. Specific conditions were determined that selectively cleaved each bond type without cleaving other bonds. These cleavages were then applied to sequence analysis of several proteins.
This document describes a biomimetic approach to synthesizing the tricyclic core structure of xyloketals. The key steps involve an ortho ester Claisen rearrangement of a chromenol to form a rearranged ester, followed by an intramolecular cationic cyclization to form the tricyclic ketal ring system. This strategy was applied to synthesize alboatrin in high yields through a short and stereocontrolled route. During the synthesis, an unexpected epi-to-natural isomerization was observed, further improving the overall yield.
The document summarizes fatty acid synthesis. It occurs in the cytoplasm of liver, adipose tissue, and mammary glands. Acetyl-CoA and malonyl-CoA serve as substrates. A fatty acid synthase complex containing multiple enzymes carries out the reactions, elongating the fatty acid chain in cycles that each add two carbon units using NADPH as a reducing agent. The process is regulated by enzymes like acetyl-CoA carboxylase.
This document provides an overview of homogeneous catalysis and biocatalysis. It discusses various homogeneous catalysts including Wilkinson's catalyst, Ziegler-Natta catalysts, and catalysts used in hydrogenation and hydroformylation reactions. It also discusses the use of enzymes in organic synthesis, including hydrolysis reactions and the synthesis of tartaric acids. Finally, it covers immobilized enzymes and various methods for enzyme immobilization.
The document summarizes a study that developed a free energy map for the co-oligomerization of HCN and NH3 in aqueous solution. Key findings include:
1) The majority of transition states favored an 8-centered, proton-transferring ring. Formamide was found to be the thermodynamic sink, while formic acid was second lowest.
2) The most thermodynamically and kinetically favorable routes from HCN to formamide coincided with HCN converting to formamidic acid and then formamide.
3) HCN, formamidic acid and formamide can produce stable 6-membered ring trimers, with formamide being the most stable product
J. Electrochem. Soc.-2003-Frank-C244-50Aaron Frank
This document analyzes the decomposition of sulfopropyl sulfonate (SPS), an additive used in acid copper electroplating. Through various analytical techniques, the researchers determined that the primary decomposition product of SPS is a thiolsulfonate. While hydrogen peroxide can oxidize SPS to form this product, experiments showed oxygen reduction on copper forms water, not peroxide. The data suggests SPS stabilizes Cu(I) and this complex is the intermediate; oxygen then reacts with the SPS/Cu(I) complex to form the thiolsulfonate decomposition product and Cu(II). Understanding this decomposition mechanism could help extend the lifespan of copper plating baths.
The peptide bond forms spontaneously via a condensation reaction that releases a small molecule like water. It has partial double bond character that restricts rotation, resulting in planar geometry. The phi and psi angles of the peptide backbone determine its conformation within allowed regions of the Ramachandran plot, avoiding steric clashes. While most peptide bonds adopt the trans configuration, proline favors cis. Protein function arises from its precise three-dimensional structure.
This document reports on a study investigating the free radical reaction between alkanes and carbon tetrachloride in solution. Product studies and kinetic electron paramagnetic resonance methods were used. The following key points are made:
1) Trichloromethyl radicals abstracted hydrogen from simple alkanes like cyclopentane and cyclohexane with rate constants of around 60 M-1s-1 in solution, in good agreement with gas phase data.
2) However, rate constants for chlorine abstraction by alkyl radicals from carbon tetrachloride were around 104 M-1s-1 in solution, around two orders of magnitude higher than in the gas phase.
3) Possibilities for this effect
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
The document describes using reversible addition-fragmentation chain transfer (RAFT) polymerization to synthesize novel block copolymers containing both a polyolefin block and a poly(styrene-co-maleic anhydride) block. Specifically, it details:
1) Using a commercially available polyolefin (Kraton L-1203) modified with a dithioester group to serve as a macroinitiator for RAFT polymerization and form the polyolefin block.
2) Conducting RAFT polymerizations of styrene and styrene-co-maleic anhydride using this macroinitiator and a small molecule RAFT agent to form the second block and yield polyolefin
This document describes the total synthesis of 2,6-dideoxy-2,6-imino-7-O-β-D-glucopyranosyl-D-glycero-L-gulo-heptitol hydrochloride (8), a potent inhibitor of α-glucosidases. The key steps involve homologation of D-glucose to form the aglycon, amination to replace the ring oxygen, and glycosylation to attach a glucose moiety. The stereoselective cyclization of an intermediate sets the stereochemistry of the ring junctions. Compound 8 inhibits intestinal sucrase and other glucosidases and lowers postprandial blood glucose in
This document summarizes a study that investigated the effects of water content and temperature on the reaction mechanism and crystal properties of paracetamol produced via reactive crystallization. The researchers first optimized the solubility of paracetamol by examining mixtures of acetic acid and water at different ratios and temperatures. They found the highest solubility occurred with a 7:3 ratio of acetic acid to water at 70°C. Based on this, they determined suitable amounts of reactants for the reaction. Experiments then examined how varying the water content (0, 10g, 20g) or temperature (50-80°C) impacted the reaction kinetics, products, and crystal properties. They found paracetamol form I crystals with 99
1) An efficient protocol has been developed for the synthesis of biaryls via Pd/Cu catalyzed coupling of phenylhydrazines in water without using any ligands.
2) Both Pd and Cu catalysts were found to be essential for the reaction, with Pd(TFA)2 and Cu(OAc) providing the best results.
3) A range of substituted phenylhydrazines underwent homo- and cross-coupling reactions under the optimized conditions to provide the biaryl products in good to excellent yields.
Raman study of the polarizing forces promoting catalysis in 4 chlorbenzoyl-co...John Clarkson
J. Clarkson, P.J. Touge, K.L. Taylor, D. Dunaway-Mariano & P.R. Carey, “Raman Study of the Polarizing Forces Promoting Catalysis in 4-Chlorbenzoyl-CoA Dehalogenase”, Biochemistry, 36, 10192-10199, 1997.
1) Polymeric excipients like PEG can stabilize proteins against denaturation during freezing by increasing the transfer free energy of the protein. However, these same polymers can induce phase separation in aqueous solutions.
2) During lyophilization, the concentrating effects of freezing can cause formulations to enter the two-phase region, resulting in liquid/liquid phase separation. This subjects the protein to potential partitioning between phases with different compositions.
3) Experimental studies on hemoglobin lyophilized in PEG/dextran mixtures provide evidence that phase separation during lyophilization can damage protein structure in the dried state.
This document is the user manual for the VP-DSC MicroCalorimeter. It provides specifications for the instrument, safety information, and instructions for operation. Key details include:
- The VP-DSC allows for high sensitivity measurement of heat capacity, binding thermodynamics, and kinetics.
- Safety precautions must be followed when using hazardous or volatile solutions in the tantalum cells.
- VPViewer software interfaces with Origin for instrument control and real-time data display.
- Sections provide tutorials for common experiments, calibration procedures, troubleshooting tips, and maintenance instructions.
1. The study characterized the aggregation of recombinant human Interleukin-1 receptor type II (rhuIL-1RII) using differential scanning calorimetry (DSC) and size exclusion chromatography (SEC).
2. A scan-rate dependence in the DSC experiment and a break from linearity in initial aggregation rates near the melting temperature (Tm) suggested that protein unfolding significantly contributes to the aggregation reaction pathway.
3. A mechanistic model was developed to extract meaningful thermodynamic and kinetic parameters from the irreversibly denatured aggregation process by simulating how unfolding properties could predict aggregation rates at different temperatures above and below the Tm.
This document reviews lyophilization (freeze-drying) as a method for developing solid protein pharmaceuticals. Lyophilization generates stresses that can denature proteins, including low temperature stress, freezing stresses from increased concentration and ice formation, and drying stress from removing the hydration shell. Several studies are discussed that demonstrate denaturation of specific proteins from these stresses during lyophilization and storage. The review discusses excipient protection of proteins, lyophilization cycle design, and formulation strategies to increase stability of solid protein pharmaceuticals and overcome instability issues.
This document summarizes the calculation of translational friction and intrinsic viscosity for four globular proteins (ribonuclease A, lysozyme, myoglobin, and chymotrypsinogen A) using their detailed atomic structures. The inclusion of a 0.9 Angstrom thick hydration shell around each protein allows the calculated translational friction and intrinsic viscosity to match experimental measurements. This hydration shell thickness corresponds to a hydration level of 0.3-0.4 grams of water per gram of protein, consistent with measurements from other techniques. Using detailed protein structures thus allows hydrodynamic measurements to support a unified picture of protein hydration, in contrast to earlier models that treated proteins as ellipsoids and found widely varying hydr
This document summarizes a study that investigated how different salts screen charge interactions in proteins. Specifically, it examined the effects of NaCl, guanidinium chloride, and guanidinium thiocyanate on the stability of wild-type E. coli thioredoxin and a variant. The results suggest that more denaturing salts like guanidinium chloride are more efficient at screening charge interactions than NaCl. This efficiency correlates with the salts' position in the Hofmeister series and ability to accumulate on protein surfaces. An electrostatic model was used to estimate contributions of charge interactions to stability.
The document describes an experiment measuring the static light scattering of concentrated protein solutions as a function of concentration. Specifically, it measured bovine serum albumin, ovalbumin, ovomucoid, and mixtures of these proteins up to 125 g/L, as well as chymotrypsin A at different pH levels up to 70 g/L. The measured scattering was quantitatively accounted for by an effective hard particle model, in which each protein is represented as a hard sphere and interactions are treated as hard particle repulsions and association equilibria.
This document summarizes a study that investigated the effects of two disaccharides (trehalose and sucrose) and trimethylamine N-oxide (TMAO) on amyloid-beta (Aβ) aggregation and interaction with lipid membranes. The key findings were:
1) In the absence of lipid vesicles, trehalose and sucrose delayed Aβ aggregation as measured by Thioflavin T fluorescence, but TMAO did not affect aggregation.
2) In the presence of lipid vesicles, all three osmolytes (trehalose, sucrose, TMAO) significantly attenuated dye leakage from the vesicles induced by Aβ aggregates.
3) Hydrogen exchange mass spectrometry (HX-MS) and
1) The study examines how long- and short-range electrostatic interactions affect the rheology and protein-protein interactions (PPI) of highly concentrated monoclonal antibody (mAb) solutions.
2) At high concentrations, both long- and short-range interactions contribute significantly to PPI, whereas at low concentrations only long-range interactions are important.
3) The study uses high frequency rheology, dynamic light scattering, circular dichroism, and zeta potential measurements to characterize PPI over a range of pH and ionic strengths, and develops a 3D computer model of the mAb to study charge distribution.
This chapter reviews the oxidation of methionine residues in model peptides. It discusses how neighboring amino acids can influence the oxidation pattern of methionine. For example, when a hydroxyl radical attacks Thr-Met, the neighboring threonine residue is cleaved. It also notes that the oxidation of peptides and proteins is a complex process that depends on the nature of the oxidizing species and the peptide/protein sequence and structure. Oxidation can lead to chain reactions as oxidation products themselves can initiate further oxidation remote from the initial attack site.
The document discusses several phase diagrams generated using different experimental data visualization techniques including:
1) A phase diagram of ricin toxin A-chain created using fluorescence and circular dichroism spectroscopic data showing four protein states.
2) An empirical phase diagram of the respiratory syncytial virus determined from multiple biophysical measurements across a pH range.
3) Phase diagrams of various non-viral gene delivery vehicles and proteins mapped against pH and temperature.
Basic fibroblast growth factor (bFGF) is being investigated for its ability to accelerate wound healing. Sulfated compounds like heparin enhance the stability of bFGF against thermal denaturation. To assess the effect on bFGF shelf life, formulations containing these excipients were incubated and analyzed. In the presence of sulfated compounds, precipitates formed that dissociated back to multimers with native structure, whereas without them precipitates were unfolded protein. Disulfide-linked multimers also increased in solution with sulfated compounds. Heparin stabilized bFGF structure and prevented rearrangement of disulfide bonds, indirectly promoting multimerization. However, loss of soluble bFGF monomer still
This document summarizes the use of differential scanning calorimetry (DSC) to optimize an antibody manufacturing process. DSC was used to screen conditions for a viral inactivation step and identify increased pH storage conditions for maximum stability. Low pH treatment reduced thermal stability, indicating structure loss. DSC provided insights into instability causes and process improvements, demonstrating its role in biotherapeutic development.
This document describes a study on controlled intracranial delivery of antibodies in rats. The researchers developed polymer matrices and microspheres for long-term antibody release directly in the brain. They implanted polymer discs containing IgG antibodies in rat brains and measured IgG concentrations at the implantation site and other brain regions over 28 days, finding highest levels with the polymer implants. The polymer provided sustained antibody levels beyond what was achieved with direct injection.
1) Aspartame degradation kinetics depend on factors like pH, temperature, buffer type and concentration, and water activity. Higher temperatures, pH, buffer concentrations and water activities increase degradation rates.
2) The activation energies for aspartame degradation decrease with increasing pH or moisture content. Phosphate buffer significantly enhances degradation more than citrate buffer.
3) In solid systems, degradation rates increase with higher initial buffer concentrations and water activities. However, the glass transition temperature does not influence degradation rates as much as water activity.
This chapter discusses the application of light scattering techniques to analyze the solution behavior of protein pharmaceuticals. It provides examples of using light scattering to characterize proteins and protein complexes, detect soluble aggregate formation, and elucidate protein-ligand interactions. The chapter also describes the theoretical background and instrumentation for light scattering measurements and analysis. It presents applications of light scattering including analyzing self-associating protein systems, selecting optimal solvent conditions, and studying the kinetics of molecular interactions.
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 presents a study using differential scanning calorimetry (DSC) to examine the thermal stability of S-protein and its complexes with S-peptide at pH 7.0. DSC measurements showed that S-protein denatures through a reversible two-state transition with a denaturation temperature between 38.5-40.0°C and enthalpy of 165-180 kJ/mol, demonstrating its lower stability without S-peptide. A two-dimensional nonlinear regression analysis of excess heat capacity curves at varying temperatures and S-peptide concentrations was used to determine the binding thermodynamic parameters, yielding values of Kb = 1.10 × 106 M-1, ΔbH = -185 kJ
The document summarizes the origin of photosensitivity in a monoclonal immunoglobulin G (IgG). UV irradiation of the monoclonal IgG causes a 70% decrease in intrinsic fluorescence and the appearance of new fluorescence, suggesting photooxidation of one or two tryptophan residues. This leads to extensive quenching of the protein's fluorescence through nonradiative energy transfer, even though few residues are directly oxidized. The photooxidation products, N-formylkynurenine and kynurenine, absorb light above 300 nm and contribute to changes in the UV-visible absorption spectrum with irradiation.
This study examines the effect of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of lipid bilayer membranes composed of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE), and dimyristoylphosphatidylglycerol (DMPG) using differential scanning calorimetry. The results show that GS interacts more strongly with anionic DMPG bilayers than with zwitterionic DMPC or DMPE bilayers, reducing the temperature and cooperativity of DMPG's phase transition to a greater degree. In contrast, GS has little effect on DMP
1. Proc. Natl. Acad. Sci. USA
Vol. 88, pp. 2854-2858, April 1991
Biochemistry
Calorimetric determination of the enthalpy change for the a-helix
to coil transition of an alanine peptide in water
(L-alanine helix/peptide hydrogen bond/helix-coil enthalpy)
J. MARTIN SCHOLTZ*, SUSAN MARQUSEE*t, ROBERT L. BALDWIN**, EUNICE J. YORK§, JOHN M. STEWART§,
MARCELO SANTORO¶1, AND D. WAYNE BOLEN¶
*Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305; §Department of Biochemistry, University of Colorado Health
Sciences Center, Denver, CO 80262; and IDepartment of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL 62901
Contributed by Robert L. Baldwin, December 7, 1990
ABSTRACT The enthalpy change (AM) accompanying the size and purify. Compound I represents a compromise be-
a-helix to random coil transition in water has been determined tween these conflicting considerations. Synthesis of I began
calorimetrically for a 50-residue peptide of defined sequence soon after the discovery that the sequence AEAAK, used as
that contains primarily alanine. The enthalpy of helix forma- a repeating unit, forms stable helices when there are three
tion is one of the basic parameters needed to predict thermal repeats (9). The possible ion pairs formed by the Glu-, Lys'
unfolding curves for peptide helices and it provides a starting residues with an i, i + 3 spacing make only a minor contri-
point for analysis of the peptide hydrogen bond. The experi- bution to helix stability; the major factor in helix formation is
mental uncertainty in AM reflects the fact that the transition the high helix propensity of the alanine residues (1).
curve is too broad to measure in its entirety, which precludes Calorimetric measurements of Al have been reported for
fitting the baselines directly. A lower limit for All of unfolding, a-helix formation by poly(L-glutamic acid) (10) and poly(L-
0.9 kcal/mol per residue, is given by assuming that the change lysine) (11), and noncalorimetric estimates of AH have been
in heat capacity (ACp) is zero, and allowing the baseline to reported for these and other polypeptides (ref. 12 and review
intersect the transition curve at the lowest measured Cp value. in ref. 13). The theory of Zimm and Rice (14) has been used
Use of the van't Hoff equation plus least-squares fitting to to take account of the ionization of poly(L-glutamic acid) and
determine a more probable baseline gives AH = 1.3 kcal/mol poly(L-lysine) (12, 13). These ionizable polypeptides form
per residue. Earlier studies of poly(L-lysine) and poly(L- random coils when fully ionized and often aggregate when
glutamate) have given 1.1 kcal/mol per residue. Those inves- fully uncharged; thus, monomeric helix formation can be
tigations, along with our present result, suggest that the side achieved only in conditions of partial ionization. A compar-
chain has little effect on AH. The possibility that the peptide ison of the value of AH for alanine helix formation with those
hydrogen bond shows a correspondingly large Al, and the for lysine and glutamic acid, which have long ionizable side
implications for protein stability, are discussed. chains, should reveal whether the dominant contribution to
AH is made by the a-helix backbone.
Although it has the smallest side chain except for glycine,
alanine has one of the highest helix propensities (1-4) of the
amino acids in the genetic code: alanine-rich peptides as short MATERIALS AND METHODS
as 16 residues form isolated a-helices in water (1). This fact Peptide Synthesis and Purification. Peptide synthesis was
suggests that the a-helix is an intrinsically stable structure in performed on a Biosearch 9500 automatic synthesizer with
water and that larger side chains, as well as polar side chains, stepwise solid-phase procedures (15) using a tert-butoxycar-
more often detract from helix stability than add to it. Studies bonyl (Boc)/benzyl strategy and HF cleavage. p-Methylbenz-
with model compounds (5-8) have given differing estimates hydrylamine (MBHA, polystyrene/1% divinylbenzene) resin
of the stability of the amide hydrogen bond in water but agree was used to give the C-terminal amide. Double couplings and
that competing hydrogen bonds to water drastically limit the capping by acylation with acetylimidazole were employed
stability of the amide hydrogen bond. Measurement of the routinely. A third coupling with a 1-hydroxybenzotriazole-
enthalpy change (All) and the heat capacity change (ACp) for benzotriazolyloxotris(dimethylamino)phosphonium hexaflu-
alanine helix formation should provide important information orophosphate active ester (16) was used when monitoring by
about the energetics of the peptide hydrogen bond, which is the qualitative Kaiser test showed the coupling to be incom-
one of the fundamental constants of protein stability. We plete. The synthesis was performed on a 0.4-mmol scale
expect that the temperature-independent component of AH starting with Boc-Phe-MBHA resin. The crude peptide was
should reflect the peptide hydrogen bond and van der Waals purified first by gel filtration on Sephadex G-50 in 0.1 M
contacts, and the hydrophobic interactions will be reflected acetic acid, then by reverse-phase HPLC on Vydac large-
in ACp. pore (300 A) C4 resin with gradients of acetonitrile in
We report the calorimetric measurement of AH for a-helix 0.1% trifluoroacetic acid. Amino acid composition was de-
formation by a 50-residue peptide, I, whose sequence is termined with a Beckman 6300 amino acid analyzer after
Ac-Y(AEAAKA)8F-NH2. hydrolysis for 22 hr at 1100C in 6 M HCl.
Circular Dichroism (CD) Measurements. CD spectra were
The results give the enthalpy of formation of a monomeric taken on an Aviv 6ODS spectropolarimeter equipped with a
helix of defined sequence and length. Short peptides give Hewlett-Packard 89100A temperature control unit. Cuvettes
broad thermal unfolding transition curves and small heats of with 10- or 1-mm pathlengths were employed. Ellipticity is
unfolding, whereas long polypeptides are difficult to synthe-
Abbreviations: DSC, differential scanning calorimetry; AHVH, van't
Hoff enthalpy change; AH~, calorimetric enthalpy change.
The publication costs of this article were defrayed in part by page charge tPresent address: Department of Biology, Massachusetts Institute of
payment. This article must therefore be hereby marked "advertisement" Technology, Cambridge, MA 02139.
in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.
2854
2. Biochemistry: Scholtz et al. Proc. Natl. Acad. Sci. USA 88 (1991) 2855
reported as mean molar residue ellipticity, [6] (degcm2. investigate the thermodynamics of the helix-coil transition
dmol-h), and was calibrated with (+)-10-camphorsulfonic associated with the polypeptide backbone, we desire a pep-
acid (17). CD samples were prepared by diluting aqueous tide that contains minimal side-chain interactions. This pep-
stock solutions of peptide either with 1 mM sodium citrate/1 tide appears to be well suited for this purpose.
mM sodium phosphate/i mM sodium borate containing the The peptide contains, in addition to the eight AEAAKA
indicated amount of NaCl or with 1 mM potassium phosphate repeats, blocked N- and C-terminal residues that eliminate
containing KF. In either case the pH was adjusted with HCl the unfavorable interaction of the two charged termini with
and KOH or NaOH to pH 7.0 at room temperature. Stock the helix macrodipole. The peptide also contains a single
peptide concentration was determined by measuring tyrosine tyrosine residue, so that peptide concentration can be deter-
absorbance in phosphate-buffered 6 M guanidine hydrochlo- mined accurately by tyrosine absorbance (1, 18). Peptide
ride, pH 6.0, as described (1, 18). purity was ascertained by reverse-phase HPLC on C4, C18,
Differential Scanning Calorimetry (DSC). Scanning cal- and diphenyl resins to be >95%, and amino acid analysis gave
orimetry experiments were performed in degassed, pH 7.0, the expected composition. The molecular weight of the
1.0 mM phosphate buffer containing 0.1 M NaCl. The peptide peptide was confirmed by fast atom bombardment mass
concentration in the DSC cell was 1.72 mg/ml (0.366 mM) as spectrometry [calculated (M+H)+, 4703.3; found, 4703.1
determined by tyrosine absorbance (18). The experiments gmol-1].
were performed with a MicroCal MC-2 differential scanning CD Spectra. The CD spectra of the peptide recorded at
calorimeter (MicroCal, Northampton, MA) at a scanning rate several different temperatures throughout the thermal tran-
of about 10C/min under nitrogen pressure of 30 psi (206.7 sition between helix and coil are shown in Fig. 1. The
kPa). Data analysis software was supplied by MicroCal and low-temperature spectra are typical of those found for a-he-
Ernesto Freire. For analysis, the raw data, in the form of heat lical peptides with minima at 222 and 208 nm and a maximum
flow (mcal min-1; 1 cal = 4.184 J) were converted to excess at 195 nm (19). The thermal transition shows an apparent
Cp (kcal mol - K-) by dividing each data point of the thermal isodichroic point at 202 nm, suggesting that all residues are
scan of peptide I by the scan rate (K min-1). The raw data either a-helical or random-coil, with no alternative residue
from a thermal scan of buffer vs. buffer, also corrected for conformations.
scan rate, were then subtracted. The resulting data were then CD Thermal Unfolding Curves. We measured the thermal
divided by the number of moles of I contained in the sample unfolding curves of I by CD in order to determine the curve
cell to give the excess Cp scan for peptide I. of percent helix versus temperature. This information pro-
DSC Data Analysis. Because of the broad thermal transition vides a useful check on finding the correct baselines for the
of this peptide, it is not possible to determine the pre- and DSC experiments. The thermal unfolding curves of the
posttransition baselines necessary for a complete analysis of peptide, measured by CD at 222 nm, are shown in Fig. 2 for
the calorimetric data. As a result, we are unable to determine several different peptide concentrations. The melting curves
ACp for the transition. To analyze the DSC data, we assumed are superimposable, indicating that the peptide is monomo-
ACp = 0 and employed two strategies in handling the baseline lecular at these concentrations. Data obtained by heating and
problem. cooling experiments are identical, demonstrating the revers-
The first strategy was to construct a baseline (or to "floor" ibility of the thermal transition from 0 to 70'C; however,
the data) at the lowest excess Cp appearing in the scan and
then fit the resulting set of data. This procedure provides an prolonged exposure of the peptide solution to temperatures
underestimate of the calorimetric enthalpy change (AHc,,), >70'C diminishes this reversibility. Both reversibility and
since the shape of the endotherm and the value of the van't monomolecularity are required for analysis of the thermo-
Hoff enthalpy change (AHVH) indicate that the endotherm is dynamics of the helix-coil transition.
incomplete in the temperature range covered by the DSC The difficulty of defining the CD baselines for the 100%
data. helix and 100% coiled forms of the peptide limits our analysis
The second strategy involved an attempt to estimate the of the transition. We investigated two different approaches to
baseline Cp by means of a least-squares minimization proce- solving this problem. The first was to assume that the peptide
dure. Starting with the endotherm obtained by flooring the is fully helical at 0°C, and to treat the helix-coil transition as
data to the lowest excess Cp, the baseline was systematically a two-state reaction. The observed value for [61222 at 0°C was
lowered by increments of 50 cal mol-'-K-1, and the data were then used together with a [10222 value of 0 for the fully coiled
fitted at each assumed baseline by setting ACp to zero while form of the peptide to calculate the fraction helix at each
allowing AHcw and AHVH to float. The sum of the squares of temperature lfH(l)] and AHVH. The second method used to
the residuals (SSR) for each fit was recorded for each new
baseline. The assumed Cp baseline resulting in the smallest
SSR was taken as the best estimate of the baseline, and the
corresponding values of AHVH and AHca, are reported as our
best estimates of these thermodynamic parameters.
RESULTS
Peptide Design and Synthesis. The sequence of the peptide
used in these studies, I, is based upon one of the peptides first
described by Marqusee and Baldwin (9). It is a longer version
of their (i, i + 3)E,K peptide, which contains glutamic acid
and lysine always separated by two alanine residues. There
are eight blocks of the simple repeat AEAAKA. The i, i + 3
spacing of the glutamic and lysine residues was selected 190 200 210 220 230 240 250 260
because side-chain interactions are minimal, when compared Wavelength, nm
with the i, i + 4 arrangement of the glutamic and lysine FIG. 1. CD spectra of peptide I recorded at temperatures in 20°
residues which stabilize the helix by forming intrahelical ion increments from 0C (lowest curve at 222 nm) to 800C (highest curve
pairs. These stabilizing interactions have not been demon- at 222 nm). The spectra were recorded at a peptide concentration of
strated with the (i, i + 3)E,K peptides. Since we wish to 8.50 ,uM in 1 mM potassium phosphate (pH 7.0) containing 0.1 M KF.
3. 2856 Biochemistry: Scholtz et al. Proc. Natl. Acad. Sci. USA 88 (1991)
I I Table 2. Helix-coil Al values determined from DSC data
E aa g . AAHlca, kcal mol' AHVH, kcal mol-1
E 30 -
go Sample Per peptide Per residue Per peptide Per residue
-0
1* 45.8 0.92 13.8 0.28
0)
e) 2* 40.5 0.81 14.0 0.28
~0 20 aft it 67.6 1.35 11.0 0.22
C,,
a 2t 60.0 1.20 11.3 0.23
0I 10 a
Values were obtained by fitting DSC scans to the 50-mer, assuming
x
cm
cm
10 a
C ACp = 0 while allowing AHcaj and AHVH to float.
*The baseline was imposed by "flooring" the DSC data such that the
' baseline intersects the DSC data at the point of lowest excess ACP
. 70
. ~~~(viz., at 90°C).
tThe baseline for this fitting was arrived at by truncating the data at
0 10 20 30 40
Temperature, OC
50 60 70 80
750C and successively lowering the "floored" baseline to obtain the
baseline giving a minimum in the sum of squares of residuals for
FIG. 2. Thermal unfolding curves for peptide I as monitored by fitting (see Materials and Methods). An example of the fitted data
CD. The concentrations of I for each experiment were 4.30 AM (A), is given as a solid line in Fig. 3.
8.60 gM (o), and 16.59 /AM (u). The solid poil nts represent data the most probable baseline. The baseline was successively
obtained from cooling experiments on the same saimples as indicated
above. lowered in Cp increments of 50 cal molP'K-l and a fitting
was performed for each new baseline, assuming ACp = 0 and
derive AHVH from the CD thermal unfolding curves involved allowing AHcaj and AHVH to float. When the sum of squares
a least-squares fit of the observed [61222 t( the van't Hoff of the residuals for each fitting reached a minimum, the
equation for a two-state process, allowiing temperature- baseline Cp at that minimum was taken as the most probable
dependent CD baselines for both the fully helical {[I]H(T)} baseline. Fig. 3 presents the best fit of the data, using this as
and fully coiled {[O]c(T)} forms of the pepitide. The values the most probable baseline. Table 2 provides the best-fit
calculated by the two methods are given in ITable 1. values for AHcaj and AHVH using this baseline. The AHvH
There are two primary reasons for calcul;ating AHVH. The values determined by CD (Table 1) and by DSC (Table 2)
first is to find out whether a single value of AHVH is able to agree satisfactorily, indicating that the spectroscopic and
describe the observed thermal unfolding cl irve. If the fit is calorimetric probes are monitoring the same temperature-
satisfactory, this AHVH value can be used to estimate, by dependent process.
extrapolation, the high-temperature and low'-temperature re-
gions of the curve, which cannot be measurred directly. The DSUSO
second is to compare the AHVH values calc:ulated from CD DISCUSSION
and DSC; if the two values are the same, it i implies that each Enthalpy of a-Helix Formation in Water. The alanine-based
technique is monitoring the same temperIature-dependent a-helix forms a stable structure in water because of the
process, regardless of the validity of the t%vo-state assump- favorable enthalpy of helix formation. The present study
tion. places Allcal of a-helix formation in the neighborhood of -1
DSC. The broad DSC endotherm observred for peptide I kcal per mol per residue (Table 2). This result supports the
creates a number of technical difficulties in evaluation of explanation for the high helix-forming propensity of alanine
AHvH and AHcw associated with the the rmal transition. (2) in which the a-helix backbone is stable in water.
Analysis of the thermal transition curves, the erefore, requires The broad thermal unfolding transition, observed by both
knowledge of the initial and final portions of the transition, in CD and DSC measurements, is typical for small helical
addition to information about the thermal baseline within the peptides. The broadness of the transition precludes any
transition zone. analysis of the ACP for helix formation and forces us to
We used two approaches to estimate the O baseline in the evaluate AH without the benefit of well-defined baselines.
DSC scans. The first method, in which a flat baseline was The two methods of applying baselines to the DSC scans
constructed by requiring the baseline to i intersect the en- provide practical ranges for AHal accompanying the helix-
dotherm at the lowest Cp, does a poor job if fitting the data coil transition. Simply flooring the DSC data to the smallest
at high temperatures, suggesting the endothe rm is incomplete Cp of the scan and integrating the area under the curve gives
in this temperature range (data not shown). This procedure a lower limit for AHcal of 43 kcal per mol of peptide. Use of
truncates the data, and consequently this ap proach underes- a systematic least-squares procedure for determining the
timates AHcal; values of Alcal and AHvH obt ained in this way baseline of the DSC scans gives an estimate for AHcal of 64
are given in Table 2. kcal per mol of peptide. Thus, the helix-coil AH per residue
One interpretation of the poor fit at high temperatures is is a minimum of 0.86 kcal per mol, with the best estimate of
that effects of irreversibility begin above 70)0C and might be AHcaj being around 1.3 kcal per mol per residue.
responsible for the aberrant fit. Accordingl ly, we truncated Calorimetric measurements of AH for the helix-coil tran-
the data at 750C and considered another appiroach to defining sition in water have been reported for two ionizable poly-
peptides: poly(L-glutamic acid) (10) and poly(L-lysine) (11).
Table 1. AHVH determined from CD data Both studies place AH for helix formation in the neighbor-
AHrvH, kcal mol-1 hood of -1.1 kcal per mol per residue, after correction for the
[6]H(T)* heat of ionization of the side chains. The polypeptides
Method [OIc(T)* Per pep)tide Per residue containing either L-glutamate or L-lysine form a-helices only
1 -35,200 0 11.4 0.23 under conditions of partial ionization; the fully charged forms
2 103T -40,000 -46T + 640 11.( 0.22 are not helical, whereas the uncharged forms aggregate. It is
*Values for the fully helical form, [6]H(T), and fully coiled form, only under conditions of partial ionization that either of these
[6]c(T), of the peptide are expressed in deg-cm2 -d mol1 with Tin C. polypeptides adopts a helical structure. This places restric-
The two methods used to calculate AHVH are desccribed in Materials tions on the experimental conditions available and necessi-
and Methods. tates correcting the observed AH for the ionization of the side
4. Biochemistry: Scholtz et al. Proc. NatL. Acad. Sci. USA 88 (1991) 2857
1000
750
y
Cl) 500
cw
FIG. 3. DSC scan for peptide
a)
0
I, truncated at 750C to avoid com-
x
w
plications from irreversibility. The
peptide concentration was 1.72
250 b all mg/ml in 1 mM potassium phos-
phate (pH 7.00) containing 0.1 M
NaCl. The baseline used was de-
termined using the second proce-
dure described in Materials and
Methods. The solid curve was
E, B e generated using parameters from
-50 -25 0 25 50 5 100 125 150 Table 3; AHVH = 11.3 kcal per mol
of peptide, AHcl, = 60.0 kcal per
Temperature, 0C mol of peptide.
chains. In spite of the differences between poly(L-glutamate), show stronger association and might be held together by two
poly(L-lysine), and the L-alanine-based peptide, each calori- amide hydrogen bonds, have been studied by Susi et al. (7,
metric study gives essentially the same value for the enthalpy 23). A complicating feature of the results for lactam dimers,
of helix formation in water, indicating that AH is determined as noted by Susi and Ard (23), is that "dimerization" of
chiefly by main-chain interactions. e-caprolactam is not a two-state reaction, and they suggested
The theory of the a-helix-random coil transition, which is that hydrophobic interactions might be involved in driving its
based on a statistical mechanical model for helix formation, association.
shows that the helix-coil transition is not a two-state reac- Another model compound study, which may be more
tion, even for short peptides. For a-helix formation by an applicable to strictly aqueous solution, comes from Suri et al.
infinite-chain polypeptide, the AH,,/AHVH ratio is found to (8). By measuring the second virial coefficients for interac-
be C-1/2 (20), where o-is the nucleation constant (in the range tion between several pairs of solutes in water, and by
of 10-3 to 10-4; see refs. 13 and 14) and a-1/2 can be thought interpreting the results with a group additivity principle, they
of as the length of the cooperative unit. For shorter peptides, found a significant attractive interaction between NH and CO
AHCa/AHVH is a function of chain length, and values of AHVH groups in water; they limited their studies to moderately
that are smaller than AH~a, are expected for all chain lengths. dilute solutions (<1 M) in order to obtain results character-
We have confirmed this basic prediction of helix-coil tran- istic of aqueous solution. This precaution is not feasible in the
sition theory, that AHcw >> AHVH (and therefore that a-helix other model compound studies cited above.
formation is far from being a two-state reaction), by our In addition to the model compound studies designed to
measurements of AHc, and AHVH (Table 2). estimate the strength of the amide hydrogen bond in water,
Comparison of the AH of Helix Formation with Model there have also been theoretical treatments of the problem
Compound Studies. There have been several studies of the (24, 25). In a molecular dynamics simulation of the associa-
formation in water of dimers, and possibly also higher tion of N-methylacetamide in water, Jorgensen (24) found
oligomers, of compounds such as urea (5), N-methylaceta- that association is driven by a dipolar interaction, not by
mide (6), and c-valerolactam (7) that might be held together formation of amide hydrogen bonds. Sneddon et al. (25) used
by amide hydrogen bonds. There is general agreement that a similar simulation method to study the interaction between
the dimers formed are weak and that the net strength of the two formamide molecules in water and in an apolar solvent.
amide hydrogen bond in water must be quite weak. There is They found that the hydrogen-bonded dimer is more stable in
disagreement, however, about whether specific dimer for- water than in an apolar solvent, but the Gibbs energy of amide
mation is observed at all and whether amide hydrogen bonds hydrogen bond formation in water is roughly zero. These
are responsible for dimer formation. Moreover, the model results illustrate the problems of using model compounds and
compound studies give differing estimates of AH ofthe amide studies of dimer formation to investigate the properties of the
hydrogen bond in water. amide hydrogen bond in water. In contrast, peptide a-helix
Schellman (5) used accurate literature data for the heat of formation is a monomolecular reaction that can be studied in
dilution of urea solutions to show that urea dimer formation dilute solution and yields several peptide hydrogen bonds
is likely and that AH of dimer formation corresponds to -1.5 formed for each helical molecule.
kcal per mol per putative amide hydrogen bond. Schonert One particularly germane theoretical treatment of the
and Stroth (21) found that binding of urea to simple peptides stability of the a-helix in water comes from Ooi and Oobatake
has similar thermodynamic properties to the urea dimer (26). Using parameters derived from the thermodynamics of
studied by Schellman; they pointed out, however, that un- protein unfolding and from model compound studies of the
certainty in estimating small dimerization constants has a effects of water on the unfolded and folded forms of a protein,
large effect on the estimated AH. Further studies of the urea they are able to predict AH and its temperature dependence
dimer system by Barone et al. (22) gave results consistent for an isolated alanine a-helix in water. Their results suggest
with Schellman's original estimate. Dimeric lactams, which that helix formation is enthalpy-driven, and the predicted AH
5. 2858 Biochemistry: Scholtz et al. Proc. NatL. Acad. Sci. USA 88 (1991)
for unfolding of a 40-residue alanine helix (0.86 kcal per mol 1. Marqusee, S., Robbins, V. H. & Baldwin, R. L. (1989) Proc.
per residue) is close to the AHcw we observe for our peptide. Natl. Acad. Sci. USA 86, 5286-5290.
They are also able to estimate the temperature dependence of 2. Padmanabhan, S., Marqusee, S., Ridgeway, T., Laue, T. M. &
AH (that is, ACp). They predict that ACp is small (2.0 cal per Baldwin, R. L. (1990) Nature (London) 344, 268-270.
mol of residue per K), and that the major contribution to the 3. Ihara, S., Ooi, T. & Takahashi, S. (1982) Biopolymers 21,
large AH for helix formation is temperature-independent. 131-145.
4. Takahashi, S., Kim, E.-H., Hibino, T. & Ooi, T. (1989)
Their work suggests that hydrogen bond formation and van Biopolymers 28, 995-1009.
der Waals contacts should be the dominant factors stabilizing 5. Schellman, J. A. (1955) C. R. Trav. Lab. Carlsberg 29, 223-
the a-helix. Further work is needed to test this prediction. 229.
Implications for Protein Folding and Structure. One of the 6. Klotz, I. M. & Franzen, J. S. (1962) J. Am. Chem. Soc. 84,
fundamental constants of protein stability is the strength of 3461-3466.
the peptide hydrogen bond in water. For a typical globular 7. Susi, H., Timasheff, S. N. & Ard, J. S. (1964) J. Biol. Chem.
protein, the majority of the backbone amides are involved in 239, 3051-3054.
hydrogen-bonding interactions in the folded protein, and 8. Suri, S. K., Spitzer, J. J., Wood, R. H., Abel, E. G. & Thomp-
most of these amides are fully solvated in the unfolded son, P. T. (1985) J. Solution Chem. 14, 781-794.
9. Marqusee, S. & Baldwin, R. L. (1987) Proc. Nat!. Acad. Sci.
protein. Our results place Al for helix formation in the USA 84, 8898-8902.
neighborhood of -1 kcal per mol per residue. It appears that 10. Rialdi, G. & Hermans, J. (1966) J. Am. Chem. Soc. 88,
the major contribution to this AH comes from the peptide 5719-5720.
backbone itself, which suggests the enthalpy of the peptide 11. Chou, P. Y. & Scheraga, H. A. (1971) Biopolymers 10, 657-
hydrogen bond is substantial. Even if this enthalpic contri- 680.
bution to the stability of the folded protein is unique to the 12. Hermans, J. (1966) J. Phys. Chem. 70, 510-515.
a-helix, -1 kcal per mol per residue would still represent a 13. Ptitsyn, 0. B. (1972) Pure Appl. Chem. 31, 227-244.
major factor in the stability of protein structure. Further work 14. Zimm, B. H. & Rice, S. A. (1960) Mol. Phys. 3, 391-407.
is required to analyze the factors contributing to AH for 15. Stewart, J. M. & Young, J. D. (1984) Solid Phase Peptide
a-helix formation and to determine whether the peptide Synthesis (Pierce, Rockford, IL).
16. Hudson, D. (1988) J. Org. Chem. 53, 617-624.
hydrogen bonds found in 8-sheets and p-turns also show 17. Chen, G. C. & Yang, J. T. (1977) Anal. Lett. 10, 1195-1207.
large values of AH. 18. Brandts, J. F. & Kaplan, L. J. (1973) Biochemistry 12, 2011-
2024.
We thank the National Institutes of Health Clinical Mass Spec- 19. Woody, R. W. (1985) in The Peptides, eds. Udenfriend, S.,
trometry Resource, University of Colorado, supported by Grant Meienhofer, J. & Hruby, J. R. (Academic, New York), Vol. 7,
RRO1152, and the Massachusetts Institute of Technology Mass pp. 15-114.
Spectrometry Facility, supported by National Institutes of Health 20. Applequist, J. (1963) J. Chem. Phys. 38, 934-941.
Center for Research Resources Grant RR00316, for the fast atom 21. Schonert, H. & Stroth, L. (1981) Biopolymers 20, 817-831.
bombardment mass spectra. We thank Robert J. Binard for amino 22. Barone, G., Castronuovo, G., Del Vecchio, P. & Giancola, C.
acid analysis and Doug Barrick, Hong Qian, and John Schellman for (1989) J. Chem. Soc. Faraday Trans. 1 85, 2087-2097.
critical review of the manuscript. J. M. Scholtz is a Public Health 23. Susi, H. & Ard, J. S. (1969) J. Phys. Chem. 73, 2240-2241.
Service Postdoctoral Fellow (GM13451). S.M. acknowledges sup- 24. Jorgensen, W. L. (1989) J. Am. Chem. Soc. 111, 3370-3371.
port from the National Institutes of Health Medical Scientist Training 25. Sneddon, S. F., Tobias, D. J. & Brooks, C. L. (1989) J. Mol.
Program (GM7365). This work was supported by grants from the Biol. 209, 817-820.
National Institutes of Health (GM31475) and National Science Foun- 26. Ooi, T. & Oobatake, M. (1991) Proc. Natl. Acad. Sci. USA 88,
dation (DMB 8904394) to M.S. and D.W.B. 2859-2863.