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.
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.
The document examines the proteomic response of Saccharomyces cerevisiae to hydrogen peroxide induced oxidative stress. Twelve protein spots were analyzed using mass spectrometry, with one spot identified as the protein Sah1. Sah1 is involved in the transmethylation metabolic pathway, which produces glutathione to relieve oxidative stress. Exposure to hydrogen peroxide increased the levels of Sah1, likely to increase glutathione production and combat the reactive oxygen species. Proteomics allows the study of entire protein networks and interactions, helping uncover proteins' functions during stress responses.
A Novel Statistical Method for Thermostable Proteins Discrimination IJORCS
This document summarizes a novel statistical method for discriminating between thermostable proteins. The method uses amino acid frequency, dipeptide frequency, and physical-chemical features extracted from protein sequences to classify proteins as mesophilic, thermophilic, or hyper-thermophilic. The researchers tested the effect of each set of features individually and combined on classification accuracy. Their results show the proposed method using all feature types together achieved the best performance, correctly classifying over 90% of proteins on average.
Year 12 biology early comm presentation intro onlyRachelCaico
This document contains information for students taking an early commencement biology course, including:
1. An outline of the contents of the course handbook, which includes the textbook, worksheets, presentations, and assessment information.
2. Details of the two biology exams for Units 3 and 4, which will assess students' understanding of key knowledge and skills through a series of questions.
3. An overview of the two areas of study in Unit 3 - Molecules of Life and Detecting & Responding. It lists the key topics to be covered and outcomes to be assessed for each area of study.
4. Details of the school-assessed coursework and examination that make up the 17
The document discusses lipid peroxidation in seeds and its effects. It defines lipid peroxidation as the oxidative degradation of lipids caused by reactive oxygen species that damage cell membranes. Lipid peroxidation occurs through initiation, propagation, and termination steps and produces reactive aldehydes. It is a major cause of seed deterioration that damages membranes and DNA. Levels of lipid peroxidation enzymes like lipoxygenases correlate with seed storability, with "good storers" having lower lipoxygenase activity. The document also describes methods to measure lipid peroxidation products.
This document discusses protecting groups in organic chemistry. Protecting groups are atoms or groups that are temporarily attached to functional groups to prevent undesirable side reactions from occurring. The document defines protecting groups and lists advantages and disadvantages. It provides examples of common protecting groups for alcohols, amines, carbonyls, and carboxylic acids. Guidelines for selecting protecting groups include being inert, easily removed, allowing desired reactions while preventing undesired ones, not introducing new stereocenters, and being easily and selectively removed under mild conditions. References for further information are provided.
This document discusses protein structure and denaturation. It explains that protein structure is stabilized by covalent and non-covalent bonds. Denaturation is the loss of a protein's native structure, resulting in changes to its physical, chemical, and biological properties. Denaturation can be caused by physical agents like heat or chemicals agents like acids. Characteristics of denatured proteins include losing biological activity and becoming insoluble in their original solvent. Denaturation is usually irreversible but some proteins can renature by removing the denaturing agent.
The document discusses reactive oxygen species (ROS) and antioxidants. ROS such as superoxide, hydrogen peroxide, and hydroxyl radicals are produced through normal cellular processes but can cause tissue damage if levels become too high. Antioxidants help prevent this damage by neutralizing ROS. The document outlines various antioxidants like vitamins C and E, carotenoids, polyphenols, glutathione, and superoxide dismutase. It also describes how ROS can damage proteins, lipids, and DNA and discusses the role of antioxidants and ROS in periodontal disease pathogenesis.
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.
The document examines the proteomic response of Saccharomyces cerevisiae to hydrogen peroxide induced oxidative stress. Twelve protein spots were analyzed using mass spectrometry, with one spot identified as the protein Sah1. Sah1 is involved in the transmethylation metabolic pathway, which produces glutathione to relieve oxidative stress. Exposure to hydrogen peroxide increased the levels of Sah1, likely to increase glutathione production and combat the reactive oxygen species. Proteomics allows the study of entire protein networks and interactions, helping uncover proteins' functions during stress responses.
A Novel Statistical Method for Thermostable Proteins Discrimination IJORCS
This document summarizes a novel statistical method for discriminating between thermostable proteins. The method uses amino acid frequency, dipeptide frequency, and physical-chemical features extracted from protein sequences to classify proteins as mesophilic, thermophilic, or hyper-thermophilic. The researchers tested the effect of each set of features individually and combined on classification accuracy. Their results show the proposed method using all feature types together achieved the best performance, correctly classifying over 90% of proteins on average.
Year 12 biology early comm presentation intro onlyRachelCaico
This document contains information for students taking an early commencement biology course, including:
1. An outline of the contents of the course handbook, which includes the textbook, worksheets, presentations, and assessment information.
2. Details of the two biology exams for Units 3 and 4, which will assess students' understanding of key knowledge and skills through a series of questions.
3. An overview of the two areas of study in Unit 3 - Molecules of Life and Detecting & Responding. It lists the key topics to be covered and outcomes to be assessed for each area of study.
4. Details of the school-assessed coursework and examination that make up the 17
The document discusses lipid peroxidation in seeds and its effects. It defines lipid peroxidation as the oxidative degradation of lipids caused by reactive oxygen species that damage cell membranes. Lipid peroxidation occurs through initiation, propagation, and termination steps and produces reactive aldehydes. It is a major cause of seed deterioration that damages membranes and DNA. Levels of lipid peroxidation enzymes like lipoxygenases correlate with seed storability, with "good storers" having lower lipoxygenase activity. The document also describes methods to measure lipid peroxidation products.
This document discusses protecting groups in organic chemistry. Protecting groups are atoms or groups that are temporarily attached to functional groups to prevent undesirable side reactions from occurring. The document defines protecting groups and lists advantages and disadvantages. It provides examples of common protecting groups for alcohols, amines, carbonyls, and carboxylic acids. Guidelines for selecting protecting groups include being inert, easily removed, allowing desired reactions while preventing undesired ones, not introducing new stereocenters, and being easily and selectively removed under mild conditions. References for further information are provided.
This document discusses protein structure and denaturation. It explains that protein structure is stabilized by covalent and non-covalent bonds. Denaturation is the loss of a protein's native structure, resulting in changes to its physical, chemical, and biological properties. Denaturation can be caused by physical agents like heat or chemicals agents like acids. Characteristics of denatured proteins include losing biological activity and becoming insoluble in their original solvent. Denaturation is usually irreversible but some proteins can renature by removing the denaturing agent.
The document discusses reactive oxygen species (ROS) and antioxidants. ROS such as superoxide, hydrogen peroxide, and hydroxyl radicals are produced through normal cellular processes but can cause tissue damage if levels become too high. Antioxidants help prevent this damage by neutralizing ROS. The document outlines various antioxidants like vitamins C and E, carotenoids, polyphenols, glutathione, and superoxide dismutase. It also describes how ROS can damage proteins, lipids, and DNA and discusses the role of antioxidants and ROS in periodontal disease pathogenesis.
This document summarizes Christopher Fenoli's research on the synthesis of novel trithiocarbonate monomers and their application in covalent adaptable networks (CANs). Specifically, it discusses the development of a facile synthetic approach to create trithiocarbonate and allyl sulfide monomers, exploration of how monomer structure affects stress relaxation in polymer networks, and implementation of the new monomers in photo-responsive and stimuli-responsive polymer networks for applications like on-demand adhesives.
This document summarizes a study on synthesizing monodisperse mesoporous TiO2 spheres with tunable sizes between 0.6 and 3.1 μm. The key findings are:
1) Increasing the reaction temperature or Ti source (titanium isopropoxide) concentration results in smaller sphere sizes with lower monodispersity.
2) Using purified titanium isopropoxide and n-dodecylamine as a surfactant leads to the largest spheres sizes with good monodispersity.
3) Decreasing the water or titanium isopropoxide concentration in the reaction increases the sphere size but can decrease monodispersity.
Title: Antioxidants for vitiligo and photoprotection: new insights and possible therapeutic implications.
Introduction: It is known that oxidative stress is of main importance in the pathogenesis of vitiligo and in the cutaneous photo-damage. Reactive oxygen species (ROS) can start a pathogenetic cascade that leads to photo-carcinogenesis and photo-aging. Indeed, ROS induce DNA oxidative damage (i.e., strands breaks, nucleic acids oxidations, mutagenic lesions), proteins and lipid peroxidation and dermal accumulation. An imbalance in ROS scavenging systems have been reported in vitiligo patients, and hydrogen peroxide excess is known to induce melanocytes damage. Hence, the use of antioxidants (AOs) in dermatology, both endogenous and exogenous, have been investigated during the last two decades.
Aims & Scope: To evaluate the current knowledge about the use of AOs for increase photo-protection and vitiligo prevention and/or treatment.
Material and Methods: Antioxidants can be administered both topically and orally: the pros and cons of each one of these ways is discussed. 13 AOs have been chosen on the basis of their beneficial effects for photo-protection and vitiligo, and described in details. For each AO agent, the most recent and important studies have been reported. The Exogenous AOs molecules here discussed include: vitamin C, vitamin E, vitamin A, 4 flavoinoids (i.e., resveratrol, quercetin, tea polyphenols and soy isoflavones) Polypodium leucotomos extract, curcumin, capsaicin. Endogenous AOs include: polyunsatured fatty acids, glutathione, melatonin.
Results: Many recent studies on AOs show promising results, in both topical and oral administration of various antioxidants agents, alone or in combination with other AOs, minerals or aminoacids, and with phototherapy (i.e., narrow band UVB or PUVA). However, large part of the last decades’ studies have used in vitro models or animal models (especially mice), while few investigation have involved human beings.
Comments: Further investigations on human model, including randomized double-blind trials on large population groups, are necessary to obtain statistically significant data and to test different treatment modalities (oral vs topical, alone vs combined) of the various AOs molecules.
2 questions with 3 multiple choice:
1) Which of the following AOs have shown to protect against several cancers, beside skin cancer?
A. Vitamin C
B. Cathechins
C. Genstein
2) Which of the following AOs combination has been recently demonstrated toimprove the resistance of cultured cells to oxidative stress, in vitro?
A. Resveratol and capsaicin
B. Curcumin snd capsaicin
C. Curcumin and Polipodyum leucotmos exract
- DISCLAIMER-
This PPT is loaded as student material "as is", from the VRF Vitiligo Master Class Barcelona November 2011; VRF does not endorse or otherwise approve it.
This document discusses reactive oxygen species (ROS) and their role in periodontal tissue damage. It begins with an introduction to periodontal diseases and defines ROS and free radicals. It describes the various ROS like superoxide, hydrogen peroxide, hydroxyl radicals, and lists sources of free radicals. Oxidative stress is defined. Mechanisms of tissue injury caused by ROS affecting proteins, lipids, DNA are outlined. Methods to measure ROS and oxidative damage in biological samples are presented. The role of ROS in periodontal tissue damage is discussed based on Halliwell's postulates. Several studies measuring local ROS in periodontitis are summarized. The antioxidant defense system and various antioxidants like vitamin C, vitamin E, carotenoids,
Free radicals are highly unstable chemical species with unpaired electrons that can damage cells. Reactive oxygen and nitrogen species are important free radicals generated through normal cellular processes and environmental exposures that can initiate chain reactions. Free radicals attack and degrade membranes, proteins, and nucleic acids. This can lead to lipid peroxidation, protein oxidation, and DNA damage implicated in various diseases. Cells employ antioxidant defenses and enzymes like catalase and superoxide dismutase to limit free radical damage.
INTRODUCTION
WHAT IS DENATURATION?
SOME EXAMPLES OF DENATURATION
THERMAL DENATURATION.
HOW DENATURATION OCCURES AT THE LEVEL OF PROTEIN STRUCTURE
NUCLIEC ACID DENATURATION
DENATURANTS
CONSEQUENSES OF DENATURATION
CONCLUSION
REFERENSE
The document discusses various aspects of antioxidants and screening models used to evaluate antioxidant potential. It introduces free radicals and their sources, types including superoxide, hydroxyl and nitric oxide radicals. Various diseases associated with oxidative stress are mentioned. Different in vitro screening models to test antioxidant capacity against reactive oxygen and nitrogen species are described, including DPPH, ABTS, FRAP, ORAC assays. Natural sources of antioxidants from plants used in Ayurveda and their potential is highlighted.
The study aimed to detect and analyze novel diterpenoid dioxygenase genes (ditA1) involved in the degradation of resin acids, which are naturally produced by trees and released during wood pulping processes. Using newly designed PCR primers, ditA1 homolog genes were amplified from various Pseudomonas, Burkholderia, and Cupriavidus strains. All isolates containing a ditA1 homolog could grow on dehydroabietic acid and expressed ditA1 constitutively or in response to dehydroabietic acid, demonstrating their role in degradation. Evolutionary analyses indicate ditA1 and gyrB genes have coevolved from ancestral variants in Pseudomonas, Burkholderia, and Cupriavid
The document discusses antioxidants and their role in human health. It defines antioxidants as molecules that inhibit the oxidation of other molecules and prevent free radical damage. Free radicals are unstable molecules that can damage cells, but antioxidant enzymes work to stabilize free radicals. Many health conditions are linked to oxidative stress caused by free radicals, including aging, cancer, diabetes and neurodegenerative diseases. The body has both endogenous and dietary antioxidant systems to protect against free radical damage through catalytic removal of radicals and protection of macromolecules.
This document discusses protein denaturation. It defines protein denaturation as the alteration of a protein's native tertiary and quaternary structure due to external stresses like heat, chemicals, or pressure. This disrupts bonds within the protein and can disrupt cell activity or cause cell death. Protein denaturation can be reversible or irreversible depending on if the protein can regain its native structure. Common causes of denaturation include temperature changes, pH changes, oxidation, and enzymatic degradation. Effects include loss of biological activity and changes to size, shape, and physical appearance.
In this ppt, we will explane what is the antioxidant and what is the free radical and also, we ex plane the mode of action for the poth.
Also we have mentioned the evaluation of antioxidant . And mentioned the drug candidate and other uses like anti aging.
I think it is important subject, read it and if there any notes please tell us .
thanks, enjoy^_^
1. Polymers are large macromolecules formed by chemical bonding of repeating structural units called monomers.
2. Polymers can be classified based on their source, structure, intermolecular forces, process of polymerization, types of monomers, and biodegradability.
3. Common natural polymers include rubber from plants and silk/wool from animals, while synthetic polymers are man-made like nylon, polyester, and neoprene. Semisynthetic polymers are derived from natural polymers like rayon.
Use RT2 Profiler PCR Arrays to profile gene expression of key
regulators and effectors of necrosis, apoptosis, and autophagy pathways in
cells treated with oxidative stress inducers vs untreated controls.
Compare expression profiles to identify differentially expressed genes that
may determine cell fate decision under oxidative stress.
Proline is an imino acid that accumulates in plants under stress conditions like drought, salt, temperature extremes, and heavy metals. It has multiple protective functions including acting as an osmolyte, maintaining redox balance, and protecting proteins and membranes from oxidative damage. Proline is synthesized through the glutamate and ornithine pathways and regulated by enzymes like P5CS and P5CR. Accumulation of proline helps improve stress tolerance by enhancing antioxidant enzymes and reducing reactive oxygen species levels.
Carbon can form diverse and complex molecules and is the main component of biological molecules besides water. It can bond up to four other atoms in chains, branches and rings to form the carbon skeletons of organic compounds. The number and arrangement of functional groups like hydroxyl, carbonyl, carboxyl, amino and phosphate groups attached to these carbon skeletons determine each molecule's unique properties and ability to undergo chemical reactions. Isomers are compounds with the same molecular formula but different structures or spatial arrangements that can have different properties.
This document discusses protein denaturation, which is defined as any change that alters a protein's unique 3D structure without breaking peptide bonds. Denaturation can be caused by heat, acids, bases, detergents, or other physical and chemical factors. It disrupts secondary, tertiary, and quaternary protein structures but not the primary amino acid sequence. Denatured proteins lose biological activity and may aggregate. In some cases denaturation can be reversed but often it is permanent.
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,
Vitamin E has four types - alpha, beta, gamma, and delta - that differ based on their methyl group position. Alpha-tocopherol has the highest biological activity due to its antioxidant properties. Vitamin E acts as an antioxidant by removing free radicals and preventing lipid peroxidation chains reactions in cell membranes. It protects membranes by reacting with lipid peroxide radicals before they can damage polyunsaturated fatty acids. Deficiencies can cause muscular dystrophy, hemolytic anemia, and hepatic necrosis by increasing oxidative damage to tissues. Vitamin E has clinical uses for conditions like nocturnal muscle cramps, intermittent claudication, and fibrocystic breast disease.
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 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.
This document summarizes Christopher Fenoli's research on the synthesis of novel trithiocarbonate monomers and their application in covalent adaptable networks (CANs). Specifically, it discusses the development of a facile synthetic approach to create trithiocarbonate and allyl sulfide monomers, exploration of how monomer structure affects stress relaxation in polymer networks, and implementation of the new monomers in photo-responsive and stimuli-responsive polymer networks for applications like on-demand adhesives.
This document summarizes a study on synthesizing monodisperse mesoporous TiO2 spheres with tunable sizes between 0.6 and 3.1 μm. The key findings are:
1) Increasing the reaction temperature or Ti source (titanium isopropoxide) concentration results in smaller sphere sizes with lower monodispersity.
2) Using purified titanium isopropoxide and n-dodecylamine as a surfactant leads to the largest spheres sizes with good monodispersity.
3) Decreasing the water or titanium isopropoxide concentration in the reaction increases the sphere size but can decrease monodispersity.
Title: Antioxidants for vitiligo and photoprotection: new insights and possible therapeutic implications.
Introduction: It is known that oxidative stress is of main importance in the pathogenesis of vitiligo and in the cutaneous photo-damage. Reactive oxygen species (ROS) can start a pathogenetic cascade that leads to photo-carcinogenesis and photo-aging. Indeed, ROS induce DNA oxidative damage (i.e., strands breaks, nucleic acids oxidations, mutagenic lesions), proteins and lipid peroxidation and dermal accumulation. An imbalance in ROS scavenging systems have been reported in vitiligo patients, and hydrogen peroxide excess is known to induce melanocytes damage. Hence, the use of antioxidants (AOs) in dermatology, both endogenous and exogenous, have been investigated during the last two decades.
Aims & Scope: To evaluate the current knowledge about the use of AOs for increase photo-protection and vitiligo prevention and/or treatment.
Material and Methods: Antioxidants can be administered both topically and orally: the pros and cons of each one of these ways is discussed. 13 AOs have been chosen on the basis of their beneficial effects for photo-protection and vitiligo, and described in details. For each AO agent, the most recent and important studies have been reported. The Exogenous AOs molecules here discussed include: vitamin C, vitamin E, vitamin A, 4 flavoinoids (i.e., resveratrol, quercetin, tea polyphenols and soy isoflavones) Polypodium leucotomos extract, curcumin, capsaicin. Endogenous AOs include: polyunsatured fatty acids, glutathione, melatonin.
Results: Many recent studies on AOs show promising results, in both topical and oral administration of various antioxidants agents, alone or in combination with other AOs, minerals or aminoacids, and with phototherapy (i.e., narrow band UVB or PUVA). However, large part of the last decades’ studies have used in vitro models or animal models (especially mice), while few investigation have involved human beings.
Comments: Further investigations on human model, including randomized double-blind trials on large population groups, are necessary to obtain statistically significant data and to test different treatment modalities (oral vs topical, alone vs combined) of the various AOs molecules.
2 questions with 3 multiple choice:
1) Which of the following AOs have shown to protect against several cancers, beside skin cancer?
A. Vitamin C
B. Cathechins
C. Genstein
2) Which of the following AOs combination has been recently demonstrated toimprove the resistance of cultured cells to oxidative stress, in vitro?
A. Resveratol and capsaicin
B. Curcumin snd capsaicin
C. Curcumin and Polipodyum leucotmos exract
- DISCLAIMER-
This PPT is loaded as student material "as is", from the VRF Vitiligo Master Class Barcelona November 2011; VRF does not endorse or otherwise approve it.
This document discusses reactive oxygen species (ROS) and their role in periodontal tissue damage. It begins with an introduction to periodontal diseases and defines ROS and free radicals. It describes the various ROS like superoxide, hydrogen peroxide, hydroxyl radicals, and lists sources of free radicals. Oxidative stress is defined. Mechanisms of tissue injury caused by ROS affecting proteins, lipids, DNA are outlined. Methods to measure ROS and oxidative damage in biological samples are presented. The role of ROS in periodontal tissue damage is discussed based on Halliwell's postulates. Several studies measuring local ROS in periodontitis are summarized. The antioxidant defense system and various antioxidants like vitamin C, vitamin E, carotenoids,
Free radicals are highly unstable chemical species with unpaired electrons that can damage cells. Reactive oxygen and nitrogen species are important free radicals generated through normal cellular processes and environmental exposures that can initiate chain reactions. Free radicals attack and degrade membranes, proteins, and nucleic acids. This can lead to lipid peroxidation, protein oxidation, and DNA damage implicated in various diseases. Cells employ antioxidant defenses and enzymes like catalase and superoxide dismutase to limit free radical damage.
INTRODUCTION
WHAT IS DENATURATION?
SOME EXAMPLES OF DENATURATION
THERMAL DENATURATION.
HOW DENATURATION OCCURES AT THE LEVEL OF PROTEIN STRUCTURE
NUCLIEC ACID DENATURATION
DENATURANTS
CONSEQUENSES OF DENATURATION
CONCLUSION
REFERENSE
The document discusses various aspects of antioxidants and screening models used to evaluate antioxidant potential. It introduces free radicals and their sources, types including superoxide, hydroxyl and nitric oxide radicals. Various diseases associated with oxidative stress are mentioned. Different in vitro screening models to test antioxidant capacity against reactive oxygen and nitrogen species are described, including DPPH, ABTS, FRAP, ORAC assays. Natural sources of antioxidants from plants used in Ayurveda and their potential is highlighted.
The study aimed to detect and analyze novel diterpenoid dioxygenase genes (ditA1) involved in the degradation of resin acids, which are naturally produced by trees and released during wood pulping processes. Using newly designed PCR primers, ditA1 homolog genes were amplified from various Pseudomonas, Burkholderia, and Cupriavidus strains. All isolates containing a ditA1 homolog could grow on dehydroabietic acid and expressed ditA1 constitutively or in response to dehydroabietic acid, demonstrating their role in degradation. Evolutionary analyses indicate ditA1 and gyrB genes have coevolved from ancestral variants in Pseudomonas, Burkholderia, and Cupriavid
The document discusses antioxidants and their role in human health. It defines antioxidants as molecules that inhibit the oxidation of other molecules and prevent free radical damage. Free radicals are unstable molecules that can damage cells, but antioxidant enzymes work to stabilize free radicals. Many health conditions are linked to oxidative stress caused by free radicals, including aging, cancer, diabetes and neurodegenerative diseases. The body has both endogenous and dietary antioxidant systems to protect against free radical damage through catalytic removal of radicals and protection of macromolecules.
This document discusses protein denaturation. It defines protein denaturation as the alteration of a protein's native tertiary and quaternary structure due to external stresses like heat, chemicals, or pressure. This disrupts bonds within the protein and can disrupt cell activity or cause cell death. Protein denaturation can be reversible or irreversible depending on if the protein can regain its native structure. Common causes of denaturation include temperature changes, pH changes, oxidation, and enzymatic degradation. Effects include loss of biological activity and changes to size, shape, and physical appearance.
In this ppt, we will explane what is the antioxidant and what is the free radical and also, we ex plane the mode of action for the poth.
Also we have mentioned the evaluation of antioxidant . And mentioned the drug candidate and other uses like anti aging.
I think it is important subject, read it and if there any notes please tell us .
thanks, enjoy^_^
1. Polymers are large macromolecules formed by chemical bonding of repeating structural units called monomers.
2. Polymers can be classified based on their source, structure, intermolecular forces, process of polymerization, types of monomers, and biodegradability.
3. Common natural polymers include rubber from plants and silk/wool from animals, while synthetic polymers are man-made like nylon, polyester, and neoprene. Semisynthetic polymers are derived from natural polymers like rayon.
Use RT2 Profiler PCR Arrays to profile gene expression of key
regulators and effectors of necrosis, apoptosis, and autophagy pathways in
cells treated with oxidative stress inducers vs untreated controls.
Compare expression profiles to identify differentially expressed genes that
may determine cell fate decision under oxidative stress.
Proline is an imino acid that accumulates in plants under stress conditions like drought, salt, temperature extremes, and heavy metals. It has multiple protective functions including acting as an osmolyte, maintaining redox balance, and protecting proteins and membranes from oxidative damage. Proline is synthesized through the glutamate and ornithine pathways and regulated by enzymes like P5CS and P5CR. Accumulation of proline helps improve stress tolerance by enhancing antioxidant enzymes and reducing reactive oxygen species levels.
Carbon can form diverse and complex molecules and is the main component of biological molecules besides water. It can bond up to four other atoms in chains, branches and rings to form the carbon skeletons of organic compounds. The number and arrangement of functional groups like hydroxyl, carbonyl, carboxyl, amino and phosphate groups attached to these carbon skeletons determine each molecule's unique properties and ability to undergo chemical reactions. Isomers are compounds with the same molecular formula but different structures or spatial arrangements that can have different properties.
This document discusses protein denaturation, which is defined as any change that alters a protein's unique 3D structure without breaking peptide bonds. Denaturation can be caused by heat, acids, bases, detergents, or other physical and chemical factors. It disrupts secondary, tertiary, and quaternary protein structures but not the primary amino acid sequence. Denatured proteins lose biological activity and may aggregate. In some cases denaturation can be reversed but often it is permanent.
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,
Vitamin E has four types - alpha, beta, gamma, and delta - that differ based on their methyl group position. Alpha-tocopherol has the highest biological activity due to its antioxidant properties. Vitamin E acts as an antioxidant by removing free radicals and preventing lipid peroxidation chains reactions in cell membranes. It protects membranes by reacting with lipid peroxide radicals before they can damage polyunsaturated fatty acids. Deficiencies can cause muscular dystrophy, hemolytic anemia, and hepatic necrosis by increasing oxidative damage to tissues. Vitamin E has clinical uses for conditions like nocturnal muscle cramps, intermittent claudication, and fibrocystic breast disease.
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 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.
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
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.
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 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.
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.
O documento explica como alterar os parâmetros A, B, C e D na função seno f(x) = A + B sen (Cx + D) afeta o gráfico. Alterar A e B muda a imagem, enquanto alterar C e D muda o domínio. Exemplos mostram como diferentes valores para esses parâmetros deslocam, estendem ou comprimem o gráfico da onda senoidal.
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.
This study examined the effects of two mitochondrial metabolites, acetyl-L-carnitine and alpha-lipoic acid, on age-related hearing loss in rats. Twenty-one aged rats were divided into three groups receiving one of the metabolites or a placebo daily for 6 weeks. Hearing was tested before and after, and tissue samples were analyzed. The control group showed expected age-related hearing decline, while treated groups experienced reduced progression. Acetyl-L-carnitine significantly improved hearing thresholds. Both metabolites reduced mitochondrial DNA damage associated with aging and hearing loss. The results suggest these compounds can delay age-related declines by protecting mitochondrial function.
1) The study investigated the effects of two mitochondrial metabolites, alpha-lipoic acid and acetyl-L-carnitine, on age-related hearing loss in rats.
2) 21 aged rats were divided into groups receiving one of the metabolites or a control, and hearing was tested before and after.
3) The treated groups experienced less progression of hearing loss compared to controls. Acetyl-L-carnitine significantly improved auditory thresholds.
Brian Juma Nyongesa is a student at Pwani University studying for a BSc in Biochemistry. He is submitting an assignment on protein denaturation for the unit "Structures of Biomolecules" taught by Dr. Ghrish Ngeny. The document discusses protein denaturation highlighting five denaturing agents and two common protein sequencing methods. It also describes the Watson and Crick secondary structural model of DNA and draws the structure of the tripeptide Cys-Pro-Met labeling the peptide bonds.
Level of structural organization of proteins in descriptionjaygawhale
Primary Structure: This is the most fundamental level and refers to the linear sequence of amino acids linked together by peptide bonds. The specific sequence of amino acids in a protein is determined by the genetic code of an organism. A change in even a single amino acid can significantly alter the protein's function.
Secondary Structure: This level describes the localized folding of the polypeptide chain due to hydrogen bonding between the carbonyl (C=O) and amino (N-H) groups of the peptide backbone. Two main types of secondary structures are alpha helices and beta sheets. These repetitive folding patterns provide stability and serve as building blocks for higher-order structures.
Tertiary Structure: This level refers to the three-dimensional arrangement of the entire polypeptide chain, including all its folds and bends. Interactions like hydrogen bonding, ionic bonds, disulfide bridges, and hydrophobic interactions determine how the secondary structures fold and assemble in space. The tertiary structure creates a unique shape for each protein, essential for its specific function.
Quaternary Structure:** This level applies only to proteins with multiple polypeptide chains. It describes how these individual polypeptide chains (each with its own tertiary structure) come together to form a functional protein complex. The interactions between these chains are similar to those seen in the tertiary structure. An example is hemoglobin, where four polypeptide chains assemble to form the oxygen-carrying molecule in red blood cells.
Understanding these levels of structural organization is crucial for comprehending protein function. The specific sequence of amino acids (primary structure) dictates how the protein folds (secondary and tertiary structures), ultimately determining its three-dimensional shape and its ability to interact with other molecules or perform its biological role.
Delving Deeper into Protein Structural Organization: Beyond the Basics
While the four-level hierarchy provides a solid foundation, protein structural organization has fascinating intricacies. Here's a closer look:
1. Primary Structure: The Blueprint in Every Bond
Amino Acid Sequence: The primary structure is the amino acid sequence, like a string of beads with unique side chains. The order and type of these amino acids (20 different types) determine the protein's potential to fold and function.Side Chain Chemistry: The side chains of amino acids have diverse chemical properties (hydrophobic, hydrophilic, charged, etc.). These properties influence how the chain folds and interacts with its environment.Disulfide Bridges: In some proteins, cysteine residues (amino acids with a sulfhydryl group) can form covalent disulfide bridges, further stabilizing the primary structure.
2. Secondary Structure: The Local Folds
Hydrogen Bonding: The key player in secondary structure formation is hydrogen bonding between the carbonyl (C=O) and amino (N-H) groups of the peptide backbo
This document provides information about enzymes, including:
1) Enzymes are specialized proteins that act as biological catalysts to increase the rate of biochemical reactions without undergoing a change themselves.
2) The International Union of Biochemistry and Molecular Biology (IUBMB) classifies and names enzymes according to the chemical reactions they catalyze.
3) There are six major classes of enzymes based on the type of chemical reaction catalyzed: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. Each class contains subclasses that provide more specificity about the reaction.
This document discusses stability problems and prevention strategies for proteins and peptides used in drug delivery systems. It describes how protein structure, including primary, secondary, tertiary, and quaternary levels can impact stability. Physical stability problems like denaturation from changes in solvent, pH, temperature and adsorption are explained. Chemical stability issues such as deamidation, oxidation, and reduction are also outlined. Methods to prevent various stability problems involving controlling solvents, pH, temperature, and use of stabilizing agents are presented.
This document discusses enzymes, including their nomenclature and classification. It begins by defining enzymes as specialized proteins that act as biological catalysts to increase the rate of biochemical reactions without undergoing a change themselves. It then covers the basic parts and classes of enzymes, explaining that the International Union of Biochemistry and Molecular Biology developed a systematic naming convention based on the reactions enzymes catalyze. It also provides examples of different enzyme name components and classifications. The document concludes by listing references used to provide this information on enzymes and nomenclature.
This document summarizes isotope effects observed in ATP synthesis catalyzed by various enzymes. Key findings include:
1) Creatine kinase loaded with the magnetic isotope 25Mg catalyzes ATP synthesis at a higher rate than when loaded with nonmagnetic 24Mg or 26Mg.
2) The rate of ATP synthesis by creatine kinase and phosphoglycerate kinase increases linearly with the fraction of 25Mg ions present.
3) Calcium and zinc isotopes also produce isotope effects, with the magnetic 43Ca and 64Zn isotopes enhancing ATP synthesis rates compared to the nonmagnetic 40Ca and 67Zn isotopes at higher ion concentrations.
4) These magnetic isotope effects were unexpected and contradicted the
This document describes research characterizing a mutant of the cytochrome P450 BM3 enzyme from Bacillus megaterium, in which an alanine residue at position 264 was mutated to glutamate (A264E mutant). Spectroscopic and biochemical analysis showed that the glutamate residue binds directly to the heme iron in the mutant enzyme, constituting a novel Cys-Fe-Glu heme ligand set. In the wild-type enzyme, a water molecule normally binds as the sixth ligand. Substrate binding to the mutant enzyme promotes displacement of water by the glutamate. The mutant enzyme retains catalytic activity despite this altered heme ligation. This research demonstrates a new heme ligation state in a cytochrome P450 enzyme.
Abstract Heavy metal pollutants are mainly derived from growing number of
anthropogenic sources. As the environmental pollution with heavy metals increases,
some new technologies are being developed, one of these being phytoremediation.
Hyperaccumulator plant varieties can be achieved by using methods of genetic
engineering. An uptake of excessive amounts of heavy metals by plants from soil
solution leads to range of interactions at cellular level which produce toxic effects
on cell metabolism in terms of enzyme activity, protein structure, mineral nutrition,
water balance, respiration and ATP content, photosynthesis, growth and morphogenesis
and formation of reactive oxygen species (ROS).On the basis of accumulation
of heavy metals plants are divided into three main types; (i) the accumulator plants,
(ii) the indicator plants, and (iii) the excluder plants. Generally, the accumulation
of heavy metals in plant organ is in series root > leaves > stem > inflorescenc >
seed. Most of plants belong to excluder group and accumulate heavy metals in
their underground parts. When roots absorb heavy metals, they accumulate primarily
in rhizodermis and cortex. In intracellular parts, highest concentration of
heavy metals is found in cell wall. Tolerance of plants against heavy metals is due
to reduced uptake of heavy metals and increased plant internal sequestration. In the
increased plant internal sequestration mechanism, plant is manifested by interaction
between a genotype and its environment. There are biochemical machineries in
plants that work for tolerance and accumulation of heavy metals. Metal transporters
are involved in metal ion homeostasis and transportation. Some amino acids and
organic acids are ligands for heavy metals and these amino acids and organic acids
play an important role in tolerance and detoxification Phytochelatins (PCs) are produced
in plants under stress of heavy metals and play role in binding heavy metals
to complexes and salts and sequestering the compounds inside the cell so that heavy
metals can not disturb the cell metabolism. The genes for phytochelatin synthesis
have been isolated and characterized. Another low molecular weight (6–7 KDa)
cysteine-rich compounds known as metallothioneins (MTs) also play an important proteins, lipids, and nucleic acids, often leading to alterations in cell structure and
mutagenesis. There are many potential sources of ROS in plants, in addition to those
that come from reactions involved in normal metabolism, such as photosynthesis
and respiration. The balance between the steady-state levels of different ROS are
determined by the interplay between different ROS-producing and ROS-scavenging
mechanisms. A variety of proteins function as scavengers of superoxide and hydrogen
peroxide. These include, among others, superoxide dismutase (SOD), catalase
(CAT), ascorbate peroxidase (APOX), glutathione reductase (GR), thioredoxin, and
the peroxiredoxin family of protein. These protein antioxidants are supplemented
with a host of non-protein scavengers, including, but not limited to, intracellular
ascorbate and glutathione. The intoxication with some heavy metals induces oxidative
stress because they are involved in several different types of ROS-generating
mechanisms.
This document provides an overview of bioinorganic chemistry. It discusses how bioinorganic chemistry involves the study of metal species in biological systems and their essential roles. Metal ions play vital roles in many biological processes, serving functions like modifying electron flow in enzymes to control reactions, binding and orienting substrates, and providing sites for redox activity. Over 50% of proteins contain metals, and metal ions are essential for about one-third of enzymes. Bioinorganic chemistry is an interdisciplinary field that has expanded our understanding of the mechanisms by which inorganic elements facilitate biochemical reactions in living organisms.
This document summarizes research testing the ability of eight diindolylmethane derivatives to induce apoptosis in murine L5178Y lymphoma cells. The derivatives were synthesized with different substituted phenyl groups attached to the methane carbon. Testing found compound 3a, with a meta-hydroxyl group, was the most active, inhibiting 93% of cell growth and inducing 71.04% apoptosis. In general, substituents able to form hydrogen bonds and in the meta position were most effective at arresting the cell cycle. The preliminary results provide insight into how the substituent and position impact potency against lymphoma cells.
1) The document reports on spectroscopic studies of the metallo-β-lactamase from Bacillus cereus 5/B/6 aimed at investigating its chemical mechanism of inhibition.
2) Previous work derived DNA and RNA aptamers that inhibit the enzyme in the nanomolar range by likely interacting with metal ions at the active site.
3) Spectroscopy results suggest the inhibitor interacts with the Zn2 site of the enzyme's active site, perturbing its coordination sphere and supporting the role of this site in catalysis.
Cross-linking is a technique used to study protein structure and interactions. It involves using bifunctional reagents containing two reactive groups to form covalent bonds between amino acid residues, either within or between proteins. This captures transient or conditional interactions and provides structural data at higher resolution than other methods. The most common cross-linking reagents react with amino acids like cysteine, tyrosine, and lysine. Cross-linking has provided important insights into protein structure-function relationships and molecular interactions.
SELF ASSEMBLY OF IONIC COMPLEMENTARY PEPTIDES & THEIR APPLICATIONS IN NANO-BI...RIJU CHANDRAN.R
This document discusses molecular self-assembly using ionic-complementary peptides and their applications in nanobiotechnology. It introduces molecular self-assembly, noting that peptides can be engineered to form stable nanostructures through non-covalent interactions. A specific ionic-complementary peptide, EAK16-II, is discussed as a model for studying peptide assembly. Several factors that can influence peptide self-assembly are also outlined, including amino acid sequence, pH, salt concentration, and time. Finally, potential applications of ionic-complementary peptides are presented, including nanofabrication, biosensing, studying protein conformational diseases, tissue engineering, and drug delivery.
Plant responses to oxidative stress can cause physiological damage at the lipid, protein, and DNA levels. Physiologically, lipid peroxidation disrupts cell membranes, protein oxidation alters structure and function, and DNA oxidation causes mutations. Biochemically, plants have developed defense mechanisms including superoxide dismutase, catalase, peroxidase, and glutathione to neutralize reactive oxygen species. Glutathione functions in redox reactions and membrane stabilization. Plants also produce antioxidants like carotenoids to counteract oxidative stress and protect cellular components from free radical damage.
2010 engineering tocopherol biosynthetic pathway in arabidopsis leavesAgrin Life
This study genetically engineered the tocopherol biosynthetic pathway in Arabidopsis thaliana by overexpressing five genes (HPPD, VTE2, VTE3, VTE1, and VTE4) involved in tocopherol production, both individually and in combinations. The results showed that elevated expression of these biosynthetic genes affected total tocopherol content and composition. Additionally, engineering the tocopherol pathway also impacted endogenous ascorbate and glutathione pools in the leaves. Further analysis found that genes in the Halliwell-Asada antioxidant cycle were upregulated. These findings provide insight into the relationship between lipid-soluble vitamin E and water-soluble antioxidants vitamin C and
This document discusses several examples of drug receptor interactions including:
1. The biotin-avidin interaction which forms the strongest known non-covalent bond between a protein and ligand.
2. The interaction between the drug trimethoprim and bacterial/mammalian dihydrofolate reductase which helps explain the drug's selectivity.
3. DNA intercalators such as proflavin and ethidium bromide which insert between DNA base pairs, unwinding the DNA helix through hydrophobic, electrostatic, and intercalative forces.
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.
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.
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.
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) Circular dichroism arises from the differential absorption of left and right circularly polarized light by chiral molecules.
2) CD spectra are more sensitive to conformational changes in proteins and nucleic acids than absorption spectra.
3) CD spectra can provide information about secondary structure in proteins.
11 20-09 mbsb single molecule techniques.pptmganguly123
This document contains over 100 images from various scientific sources related to topics in optics, microscopy, fluorescence, plasmonics, and single molecule techniques. The images depict experimental setups, microscopy images, diagrams, graphs, and other figures illustrating different scientific phenomena and techniques. Captions provide brief descriptions and citations for the source of each image.
The document discusses the benefits of meditation for reducing stress and anxiety. Regular meditation practice can help calm the mind and body by lowering heart rate and blood pressure. Studies have shown that meditating for just 10-20 minutes per day can have significant positive impacts on both mental and physical health.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive functioning. Exercise causes chemical changes in the brain that may help boost feelings of calmness, happiness and focus.
1. The document describes quantitative characterization of polymer-polymer and polymer-protein interactions using tracer sedimentation equilibrium.
2. The dependence of the thermodynamic activity coefficient on concentration was determined using two methods: power series expansion and scaled particle theory.
3. Results show BSA-BSA interactions are weaker than BSA-ficoll interactions, which are weaker than ficoll-ficoll interactions. Scaled particle theory modeling ficoll as a hard spherocylinder and BSA as a hard sphere described the experimental data well.
2. 80 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
oxidation in a given peptide or protein. It was shown that a rather nonselective
strong oxidant such as the hydroxyl radical (HO'), produced by y-irradiation of
aqueous protein solutions, will attack proteins randomly and will oxidize amino acids
based on their location within surface accessible domains of proteins (5). More
selective oxidants such as peroxides will predominantly react with methionine and
cysteine where surface exposure as well as the protonation state of cysteine will be
important parameters. Under conditions of metal-catalyzed oxidation a peroxide may
first react with a transition metal, bound to a metal-binding site of a protein, before
the resulting reactive oxygen species will predominantly attack amino acids in the
surrounding of the metal-binding site.
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In addition, chain oxidations and radical transfer reactions within the peptide or
protein will lead to even more complex oxidation patterns. For example, protein-
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
bound peroxides (6) and in particular valine hydroperoxide (7) have been identified as
important endpoints of hydroxyl radical-induced reactions. However, these hydro-
peroxides will only be true endpoints in the absence of catalytically active transition
metals which can convert hydroperoxides into highly reactive alkoxyl radicals in a
Fenton type reaction, initiating further protein damage through subsequent reactions
of the alkoxyl radicals (8, 9). Consequently, an initial oxidative event at a protein
locus can initiate protein chain oxidations (10) where oxidation products can be
expected to appear remote from the side of the initial attack. Furthermore, several
studies on electron transfer processes in proteins have now demonstrated that
intramolecular electron transfer (or hydrogen transfer) may lead to the migration of
an initial oxidation site (77). The final oxidation products of a given peptide or
protein will, therefore, be the complex result of (i) the nature of the oxidizing species,
(ii) the nature of the peptide or protein, determining sequence and higher order
structure, and (iii) the mechanism of the reaction of the oxidizing species with the
peptide or protein as it may depend on a specific peptide or protein sequence.
In pharmaceutical formulations it is generally difficult to characterize the nature
of an oxidizing species and, consequently, the mechanism of its reaction with a
peptide or protein. However, details about these mechanisms would be desirable in
order to assist in the development of stable peptide and protein formulations. Some
information about potential reactions can be obtained from the biochemical literature
where a series of proteins has been investigated with respect to metal-catalyzed
oxidation (3, 12-14), photooxidation (15-17), and (y-radiolysis (6, 10, 18-20). In the
chemical literature, we find more detailed investigations on chemical kinetics, neigh-
boring group effects, and mechanisms of oxidation of small peptides and amino acids
(or their derivatives) by various reactive oxygen species and free radicals (see below).
Ideally, we would need to characterize the chemical mechanisms of oxidation by
various reactive oxygen species or free radicals for a given peptide and then compare
the kinetic features and product distributions for the oxidation of the same peptide
within a pharmaceutical formulation. Whenever the product distributions obtained
through both approaches match there will be a probability that the underlying
reaction mechanisms occurring within a formulation have been identified. However,
this approach will not work whenever several reactive oxygen species give the same
product with a given amino acid. This will be the case, for example, for methionine
and its major oxidation product, methionine sulfoxide, or for cysteine, and its
products cystine or cysteine sulfonic acid. In such cases, we need more refined
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
3. 4. SCHONEICH ET AL. Mechanisms of Methionine Oxidation in Peptides 81
experiments in order to identify the contributing reactive intermediates. Several
scavengers can be employed which will either selectively react with one reactive
species (e.g. superoxide dismutase with superoxide) or at least will show different
kinetics in their reactions with different reactive oxygen species. Whenever rate
constants are available we can then predict the efficiency of a scavenger towards a
reactive species and compare theoretical with experimental data. In the following, we
shall discuss some selected examples for these approaches as they relate to the
oxidation of methionine in small peptides. For this purpose we shall first introduce
several important mechanistic aspects on the free radical chemistry of some model
peptides before the discussion of some pharmaceutically relevant aspects of these
Downloaded by UNIV OF PITTSBURGH on August 19, 2010 | http://pubs.acs.org
considerations will follow. The investigated peptides have been selected on the basis
of their model character and/or their presence in an oxidation sensitive domain of cal-
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
modulin which was identified to be highly oxidation labile both in vitro (21-24) and
in vivo (25). They do as yet not represent pharmaceutical examples. However, as we
will proceed from small model peptides to larger structures in future experiments,
pharmaceutically relevant peptides will become a major focus of investigation, and
the mechanisms obtained with small model peptides are likely to be an important
basis for these studies.
Oxidation of Thr-Met: The migration of radical sites
Mechanistic studies with the hydroxyl radical. When the peptide Thr-Met was
reacted with radiation chemically produced hydroxyl radicals (HO*) in anaerobic
reaction mixtures at neutral pH, a major oxidation product was acetaldehyde, derived
from the fragmentation of the Thr side chain (26). This reaction did not occur
whenever the C-terminal Met residue was omitted, as in Thr-Leu, or when Thr was
not the N-terminal amino acid, as in Gly-Thr-Met. Moreover, acetaldehyde was not
a major reaction product when Thr-Met was subjected to one-electron oxidation by
SOzi*- (26) or by triplet carboxybenzophenone (27), indicating that it was not an
initial one-electron transfer process, i.e. the formation of a sulfur radical cation,
which led to the observed product formation.
Mechanistically, the reaction was rationalized by a fast formation of a hydroxy-
sulfuranyl radical 1 upon reaction of the hydroxyl radical with the Met residue of
Thr-Met (Scheme 1). The hydroxysulfuranyl radical subsequently underwent rapid
+
formation of a three-electron-bonded intramolecular [S.N] complex 2 (reaction 1)
which was characterized by the fast method of pulse radiolysis and had a half-life for
further decomposition of t/2 = 320 ns. A n important equilibrium (reaction 2) of
complex 2 is the open chain species containing a nitrogen-centered radical cation, a
precursor for efficient heterolytic cleavage of the C - C p bond of the Thr side chain
a
leading to acetaldehyde and a proton (though some cleavage may directly occur from
the cyclic complex 2) (reaction 3).
This mechanism bears several interesting features that are important to consider
in any study on peptide oxidation. First, from the published rate constants for the
reaction of hydroxyl radicals with methionine, it was expected that a major fraction
of hydroxyl radicals (>93%) reacted with the methionine moiety of the peptide.
Nevertheless, the major final reaction product was a species containing intact Met
but a fragmented Thr residue, indicated by the high yields of acetaldehyde (for [Thr-
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
4. 82 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
Met] < 5 x 1(H M : 60% related to the initial yield of H *. Such radical or damage
O)
transfer constitutes an important mechanistic pathway frequently observed during
the oxidation of proteins.
HN CH C07
I I
CH C=0 CH
3 2
HC-HC VH 2 !
O ^ f t| *S
H H | -CH
Downloaded by UNIV OF PITTSBURGH on August 19, 2010 | http://pubs.acs.org
3
H 0 N
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
Scheme 1 H
HN- -H 0 2
I • ICH— co; (1)
CH 3C=0 CH, CH,
I / I
CH 2 HC—HC
HC-HC (2)
O H-N.*.S<^
H O H N
H —T
H . ^CH H
(3)
HN C C07
H
I I
,C=0 CH 2
H C +
V ^CH, + CH-CH=0 + H 3
/
H ^CH,
In the absence of any detailed mechanistic information, the lack of oxidized Met
residues as major products may have led to the conclusion that Met was not a target
during the oxidation. However, as demonstrated above this is not true. In fact, Met
constituted a major point of attack for the reactive oxygen species, but radical trans-
fer reactions subsequent to Met oxidation "repaired" the initial oxidation product
(the sulfuranyl radical) and the final radical damage appeared at a different locus of
the peptide. Besides for Thr-Met we could demonstrate a similar radical transfer
reaction also for Ser-Met (26). In addition, comparable radical transfer mechanisms
within some S-alkylglutathionine derivatives (28) and y-Glu-Gly-Met-Gly (29) led
to an efficient decarboxylation of the N-terminal y-Glu residue, mediated by an
initially formed hydroxy sulfuranyl radical at the Met residue.
Returning to the example of Thr-Met, there were several other mechanistically
interesting observations. First, in the presence of oxygen the yields of acetaldehyde
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
5. 4. SCHONEICH E T A L . Mechanisms of Methionine Oxidation in Peptides 83
were significantly reduced (by ca. 50%). This finding led to the hypothesis that
hydroxysulfuranyl radicals might react competitively with molecular oxygen with the
respective products entering reaction pathways different from acetaldehyde forma-
tion. Indeed, we could demonstrate that a model hydroxysulfuranyl radical does react
8
with molecular oxygen with a rate constant on the order of 1.1 x 10 M H s - l (30,
31). Since free superoxide was not an immediate product of this reaction, this
process likely involves the addition of oxygen to the hydroxysulfuranyl radical,
resulting in a peroxyl type radical (reaction 4).
R S--OH
2 + 0 2 -> HO-S(R )-00- 2 (4)
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The structures of related peroxyl radicals have subsequently been characterized by
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
low temperature ESR experiments (32). However, the rate constant for reaction 4
appears too slow in order to affect the formation of acetaldehyde in the Thr-Met
system so that the effect of oxygen on the yields of acetaldehyde should have
additional reasons which have yet to be characterized.
co 2 co 2
H N-(Thr)-CONH
3 CH H C — NH-CO-(Thr)-NH 3
/ S ..s x
H,C CHa
Secondly, when the oxidation of Thr-Met by HO* was carried out with different
concentrations of Thr-Met it was observed that increasing concentrations of Thr-
Met led to decreasing yields of acetaldehyde but increasing yields of a one-electron
oxidized sulfur species, an intermolecular sulfur radical cation complex 3 (this species
was, again, identified by pulse radiolysis). The latter exists in equilibrium 5 with the
,+
monomeric radical cation 4 (>S ) both of them being good one-electron oxidants
themselves (33). However, we note that the radical complex 3 is significantly more
stable than the monomer 4 with regard to irreversible decomposition pathways such
as deprotonation (yielding a-(alkylthio)alkyl radicals). Thus, as
[>S.-.S<]+ (3) = >S + >S'+ (4) (5)
#
already concluded from the experiments with SC>4 - and triplet carboxybenzo-
phenone, any one-electron oxidation of the Met residue of Thr-Met, yielding a sulfur
radical cation, yields acetaldehyde less efficiently or not at all (depending on the ini-
tial oxidant). Although the sulfur atom in the hydroxysulfuranyl radical 1 formally
contains a one-electron oxidized sulfur (compared to the reactant thioether), its
chemistry is interestingly quite different compared to that of another one-electron
oxidized sulfur species, the sulfur radical cation. The similarity of both may be better
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
6. 84 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
realized by considering that both the hydroxysulfuranyl radical 1 and the monomeric
radical cation 4 are related to each other in an acid-base type equilibrium (reaction 6).
Sulfur radical cations have theoretically been shown to exist in a hydrated form 4a
where the S - 0 bond dissociation energy amounts to ca. 16.8 kcal/mole (34).
Formally, species 4a has only to deprotonate in order to transform into 1, although
some electronic (and geometric) rearrangement may also be required, depending on
the actual type of the hydroxysulfuranyl radical, i.e. o, TC, or a* radical. Thus, for
Thr-Met, a simple protonation-deprotonation equilibrium such as reaction 6 may
eventually decide on the fate of the initially formed intermediate at the sulfur.
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[>S'-OH ]+ 2 (4a) = H+ + >S'-OH(l) (6)
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
Metal-catalyzed oxidation. With this mechanistic framework at hand it was now
possible to investigate the oxidation of Thr-Met by hydrogen peroxide in the
presence of transition metals. The transition metal/hydrogen peroxide system is of
great importance for pharmaceutical formulations as they often contain metal impuri-
ties through buffer contamination and traces of peroxides from sterilization proce-
dures. Moreover, peptides themselves are well suited ligands for many transition
metals so that metal chelation by peptides may easily transfer transition metals from
production lines to storage/formulation systems.
Table 1 contains a list of some key products obtained during the reaction of Thr-
Met with HO', hydrogen peroxide in the presence of ferrous iron, and hydrogen per-
oxide in the presence of ferrous EDTA (55). It becomes immediately apparent that
the incubation of hydrogen peroxide with ferrous iron alone did not result in a
product distribution which resembles the one obtained through free hydroxyl radi-
cals. Thus, the classical Fenton system does not produce free hydroxyl radicals, at
least at pH 7.5. Nevertheless we find a significant formation of Met sulfoxide. More-
over, the sulfoxide formation by the hydrogen peroxide/ferrous iron system is very
4
rapid as the reaction was completed within ca. 2 min for 5.0 x 10" M hydrogen
peroxide, 5.0 x 10-4 M ferrous iron, and 1.0 x 10-3 M Thr-Met. In contrast, the
oxidation of 1.0 x 10-3 M Thr-Met by 5.0 x 10-4 M hydrogen peroxide alone did not
Table 1: Oxidation of Thr-Met by free hydroxyl radicals and Fenton systems
a
HO' Fen/H 0 2 2
b
[Fen(EDTA)]2-/H 0 2 2
b
-Thr-Met c
63 232±57 354 ± 5 7
Thr-Met(SO) <5.1 164±15 66 ± 12
CH CHO
3 36 6±3 65 ± 5
C0 2
13.2 d
n.d. n.d.
a
Yields are from y-radiolysis employing a dose of 100 Gly (calculated from reference 26).
b c
p H 7.5; 2 x 10-3 mol dm~3 carbonate buffer. Refers to loss of Thr-Met. Estimated from
reference 36
SOURCE: Adapted from reference 35.
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
7. 4. SCHONEICH ET AL. Mechanisms of Methionine Oxidation in Peptides 85
result in any significant formation of sulfoxide within 2 minutes, demonstrating that,
although not generating hydroxyl radicals, nevertheless ferrous iron catalyzes the oxi-
dation of Thr-Met by hydrogen peroxide. Thus, impurities of transition metals can
catalyze peroxide mediated peptide oxidation via mechanisms quite different from
the classical Fenton reaction. This does not only apply to small dipeptides as we
have also observed extremely rapid oxidation of methionine-containing dodeca-
peptides (to methionine sulfoxide) by hydrogen peroxide/ferrous iron (unpublished
results). Mechanistically, we propose that hydrogen peroxide merely binds to the
transition metal, generating a complex that transfers oxygen to the Met residue. It
seems reasonable to assume that these reactions are not only catalyzed by the metal
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per se but also further accelerated by the binding of the transition metal to the
peptide, promoting an intramolecular reaction between the ferrous iron-hydrogen
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
peroxide complex and the Met residue. In fact, some evidence for the latter was
obtained when the presence of an additional His residue promoted the iron-catalyzed
oxidation of nearby Met residues (37, 38 (although these oxidations were carried out
by a rather unspecific oxidizing system consisting of either dithiothreitol or
ascorbate, ferric iron, and molecular oxygen). It is known that proteins which contain
well-defined metal binding sites are prone to site-specific oxidation by reactive
oxygen species generated within these metal-binding sites (5,12, 13). However, in
most cases these metal-binding sites require an intact secondary and tertiary
structure of the protein. Thus, specific peptides comprising only one or the other
metal-binding amino acid residue of a metal-binding site may not necessarily suffer
site-specific oxidation like the intact metal-binding domain does. Such a case was
recently observed for the metal-catalyzed oxidation of recombinant human growth
hormone (39).
Table 1 shows that the presence of EDTA did not protect Thr-Met from oxida-
tion by hydrogen peroxide/ferrous iron. However, it becomes apparent that signifi-
cant yields of acetaldehyde are formed in this system. Thus, either hydroxyl radicals
or hydroxyl radical-like species are formed in the [FeH(EDTA)] 7hydrogen peroxide
2
system. Further evidence for the presence of hydroxyl radical-like species in the
[FeH(EDTA)]2-/hydrogen peroxide system was derived from a variation of the Thr-
Met concentration. If hydroxyl radicals or alike species were formed in our systems
we would expect higher relative yields of acetaldehyde at lower Thr-Met concentra-
tions (as described above for free hydroxyl radicals). This was indeed observed,
supporting the conclusion that at least a fraction of hydroxyl radicals or alike species
are present in our [Fell(EDTA)]2-/hydrogen peroxide system, responsible for acet-
aldehyde formation. On the other hand, significant yields of Met sulfoxide are also
present after oxidation of Thr-Met by [Fell(EDTA)]27hydrogen peroxide. Earlier,
we had shown that the reaction of free hydroxyl radicals with aliphatic sulfides did
not result in sulfoxide formation, particularly in the absence of oxygen (40). When
we oxidized Thr-Met by the [ F e ( E D T A ) ] 7 h y d r o g e n peroxide system in the
n
2
additional presence of 2-propanol (a well-characterized hydroxyl radical scavenger),
we could demonstrate that the obtained product yields could not be rationalized by
competition kinetics based on a competitive reaction of hydroxyl radicals with either
Thr-Met or 2-propanol. Thus, the yields of Thr-Met sulfoxide have to be ascribed to
a reactive oxygen species being of different nature than a hydroxyl radical.
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
8. 86 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
These results have at least two major implications for pharmaceutical formula-
tions. First, the presence of a metal chelating agent such as EDTA does not necessar-
ily protect a peptide form oxidation, but any possible protection will very much
depend on the nature of the involved species (and, of course, the nature of the transi-
tion metal). Second, there may be more than one oxidizing species in an E D T A -
containing system. In the case of our model system involving Thr-Met and
n 2
[FE (EDTA)] -/hydrogen peroxide, these species may be hydroxyl radicals (respon-
sible for acetaldehyde) as well as metal-bound peroxides (responsible for Met sulf-
oxide). In the following, we have extended these studies to model peptides containing
both a Met and a His residue.
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Oxidation of His- and Met-containing peptides: Intramolecular catalysis by His
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
When several model peptides containing His and Met were subjected to oxidation by
a system consisting of dithiothreitol, ferric iron, and molecular oxygen, the major
oxidation products were the corresponding Met sulfoxide-containing peptides (37,
41). Moreover, for some peptides, the presence of His catalyzed the oxidation of
Met, indicated by higher oxidation yields of peptides containing both His and Met as
compared to peptides that contained only Met (37, 41). In the absence of E D T A ,
the sulfoxide represented ca. 67% of the overall product yield for Gly-His-Gly-Met-
Gly-Gly-Gly. In the additional presence of EDTA (or other polyaminocarboxylate
chelators), the respective yields of Met sulfoxide decreased. However, this was not
due to any particular overall protective effect of EDTA as the overall peptide degra-
dation was still significant in the present of EDTA. Rather, the EDTA-containing
system promoted the formation of several other oxidation products besides Met
sulfoxide such as 2-oxo-histidine (structure 5), of which significant yields were
formed (41). While the formation of 2-oxo-histidine could be inhibited by the addi-
tion of 2-propanol, this was not (or significantly less) the case for Met sulfoxide.
Thus, also in the dithiothreitol/ferric iron/molecular oxygen-system, the presence of
E D T A leads to the formation of different reactive oxygen species promoting a
variety of reactions with different oxidation products.
R-CO-NH CH; CO-NH-R
5
HN. NH
Oxidation of Met-Met: Diastereoselective product formation
We have discussed the importance of equilibria 5 and 6 for the actual fate of inter-
mediates formed by an initiating oxidative event at the sulfur of Met, i.e. electron
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
9. 4. SCHONEICH ETAL. 87
Mechanisms of Methionine Oxidation in Peptides
transfer vs. reaction of hydroxyl radicals. It seemed then appropriate to investigate a
peptide in which an intramolecular sulfur-sulfur interaction may lead to a preferred
formation of sulfur radical cations, favored through intramolecular stabilization of
+
these cations as [>S.S<] complexes. For Met-Met such intramolecular complexes
had been characterized by means of pulse radiolysis (42) and laser photolysis (using
triplet carboxybenzophenone) (43). When the peptide L-Met-L-Met was subjected
to one-electron oxidation by triplet carboxybenzophenone in the presence of oxygen,
we observed the formation of monosulfoxides and disulfoxides of L-Met-L-Met (44).
Most interestingly, however, we could characterize the formation of two diastereo-
meric azasulfonium derivatives (6a and 6b), different at the configuration of the
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sulfur, and present in an approximate 3:1 ratio (44). A n absolute assignment of the
observed ratio to the two species is currently in progress. These dehydro-Met-Met
Publication Date: August 1, 1997 | doi: 10.1021/bk-1997-0675.ch004
products could also be obtained through oxidation of L-Met-L-Met by I3-, though
with a clean ratio of 1:1. Thus, the one-electron transfer oxidation process displays a
measurable diastereoselectivity and it appears that sulfur-sulfur interaction, most
+
probably within an initially formed complex [>S.S<] , is an important factor in the
observed diastereoselectivity. With this observation, we can provide another inter-
esting example on how peptide sequence (and structure) can influence product
formation during peptide oxidation.
Conclusions
The present examples demonstrate the manifold of possible oxidation pathways that
can be encountered during the oxidation of peptides even when the nature of the reac-
tive species is known and the systems under study are relatively simple. In a phar-
maceutical formulation it will be considerably more difficult to characterize oxidation
mechanisms and to design stabilization strategies based on the oxidation mechanisms.
Nevertheless, with more experimental work in progress on the oxidation of more
complex peptides, we hope to achieve such a goal at some time in the future.
In Therapeutic Protein and Peptide Formulation and Delivery; Shahrokh, Z., et al.;
ACS Symposium Series; American Chemical Society: Washington, DC, 1997.
10. 88 T H E R A P E U T I C PROTEIN AND PEPTIDE F O R M U L A T I O N AND D E L I V E R Y
Acknowledgments
The research described herein was supported by the NIH (POl A G 12993-01), the
Association For International Cancer Research (AICR), an A F P E fellowship (to
F.Z.), a graduate student fellowship from R. W. Johnson, and a Self Fellowship (to
B.M.).
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