Showing papers in "Methods in Enzymology in 1977"
TL;DR: Although diethylpyrocarbonate does not always react specifically with histidyl residues in proteins, it is more selective than other acylating agents and can give useful information about the role of histidol residues in many proteins.
Abstract: Publisher Summary Diethylpyrocarbonate has been shown to react specifically or stoichiometrically with a single histidyl residue in certain proteins. In other cases, the modification of activity has been correlated with the modification of one or more histidyl residues despite the possible modification of other residues; this correlation is facilitated by the fact that hydroxylamine removes the carbethoxy group from modified histidyl residues and tyrosyl residues, but not that of modified lysyl or sulfhydryl residues. Several enzymes have been shown to be inactivated by the modification of a residue other than a histidyl residue. Thus, although diethylpyrocarbonate does not always react specifically with histidyl residues in proteins, it is more selective than other acylating agents and can give useful information about the role of histidyl residues in many proteins. This chapter describes the way optimal conditions for reaction should be determined and the way possible side reaction should be examined.
786 citations
TL;DR: This chapter discusses the photoaffinity labeling which could be used as a method that allows to unleash the reagent at a particular time and place, when the chemical affinity labeling restricts it.
Abstract: Publisher Summary This chapter discusses the photoaffinity labeling which could be used as a method that allows to unleash the reagent at a particular time and place, when the chemical affinity labeling restricts it. The chapter notes that the possibility that a labile group of appropriate reactivity cannot be incorporated into the ligand molecule without excessive disturbance of the recognition process, there are two limitations to the affinity labeling approach. The first challenge; the range of chemical reactivity of groups that can be incorporated into the ligand is limited by the fact that these groups must not react so rapidly with water that they are destroyed hydrolytically before the ligand that carries them can reach the binding site. And secondly, it is becoming clear that some biological problems require a reagent whose reactivity remains masked until the experimenter chooses to activate it. Both of the two limitations of classical chemical affinity labeling discussed above can in principle be circumvented by the use of a photogenerated reagent.
452 citations
TL;DR: This chapter presents a procedure for nonenzymic cleavage of proteins with hydroxylamine, which provides a relatively specific means of producing large peptide fragments suitable for further chemical analysis.
Abstract: Publisher Summary This chapter presents a procedure for nonenzymic cleavage of proteins with hydroxylamine, which provides a relatively specific means of producing large peptide fragments suitable for further chemical analysis. Cleavage occurs at Asn-Gly bonds and results from the tendency of the asparaginyl side chain to cyclize, forming a substituted succinimide that is susceptible to nucleophilic attack by hydroxylamine. The increased susceptibility of Asn-Gly bonds in comparison with other asparaginyl bonds may result from the greater ease with which the asparaginyl side chain can cyclize in the absence of steric hindrance imposed by a side chain on the succeeding amino acid. The extent of cleavage achieved varies with the protein; cleavage is enhanced by complete denaturation of the protein and by the use of 6 M guanidine as a solvent during hydroxylaminolysis. A low level of cleavage at Asn-X bonds has been observed in some cases, but aspartyl bonds appear to be resistant under conditions used. The infrequency of Asn-Gly bonds in most proteins results in the production of very large fragments that may overlap CNBr-produced fragments and could serve as new start points for sequential Edman degradation.
371 citations
TL;DR: The relative paucity of AspPro bonds in proteins is both a limitation and an advantage of this specific cleavage procedure as the number of fragments obtained by application of the procedure can be advantageously small, which will simplify subsequent fractionation processes.
Abstract: Specific cleavage at AspPro bonds of a protein can be effected by exposure to acid at moderate temperature for periods up to 120 hr. The effectiveness of cleavage is usually increased by incorporating a denaturing agent in the acid solution. Some AspPro bonds are resistant to cleavage probably because of retention of protein folding even under the most vigorous conditions that have been employed, and under these circumstances some nonspecific peptide bond cleavage may occur, particularly if the exposure is prolonged.
It is very likely that cleavage at AspPro bonds will occur when acid procedures are employed in peptide bond scission or peptide separation and purification. With the larger peptides produced by cyanogen bromide cleavage it has been observed that partial cleavage at AspPro near the termini of the authentic peptide may yield fragments that copurify with the larger component, thus complicating subsequent sequence determinations.13
The relative paucity of AspPro bonds in proteins is both a limitation and an advantage of this specific cleavage procedure. The limitation is that many proteins do not contain any AspPro bonds but as the AspPro bond is never abundant the number of fragments obtained by application of the procedure can be advantageously small, which will simplify subsequent fractionation processes.
308 citations
TL;DR: Nicotinic acetylcholine receptors (nAChRs) are cholinergic receptors that form ligand-gated ion channels in the plasma membranes of certain neurons and on the postsynaptic side of the neuromuscular junction.
Abstract: Publisher Summary This chapter deals with nicotinic acetylcholine receptors. Nicotinic acetylcholine receptors (nAChRs) are cholinergic receptors that form ligand-gated ion channels in the plasma membranes of certain neurons and on the postsynaptic side of the neuromuscular junction. As ionotropic receptors, nAChRs are directly linked to ion channels and do not use second messengers. These receptors differ from muscle-type receptors in subunit composition, pharmacology, and channel properties. Nicotinic receptors are also found in many invertebrate phyla. These toxins have been indispensable tools in the exploration of the acetylcholine (ACh) binding sites and in the assay of muscle-type and some neuronal-type ACh receptors. The structure of nAChRs is understood as subunit composition, primary structure, secondary and tertiary structures, and quaternary structures. It also takes into account the ACh binding sites, the channel, and the cytoplasmic domain. The channel has three tasks. It must mitigate the high-energy barrier to the translocation of an ion from one polar aqueous phase to another, through a non-polar lipid membrane; it must select among ions both by size and by charge; and it must open and close. The energy barrier is partly mitigated by the funnel shape of the channel and by its water content. The cytoplasmic domain of each subunit consists of a short loop between M1 and M2 and a long loop between M3 and M4. Phosphorylation of sites in this loop modifies the rate of desensitization and may regulate interactions of the receptor with cytoplasmic proteins.
280 citations
TL;DR: All peptides and proteins studied in the chapter can be fully reduced with a 5-20% molar excess of tributylphosphine with PBu 3, and work on highly purified preparations of soluble proteins with known structure is presented.
Abstract: Publisher Summary To study the applicability of the tributylphosphine reduction to general protein chemistry, this chapter presents work on highly purified preparations of soluble proteins with known structure. All peptides and proteins studied [(Lys δ )-vasopressin, insulin, human serum albumin, and bovine ribonuclease] in the chapter can be fully reduced with a 5-20% molar excess of tributylphosphine with PBu 3 . No strong denaturing agent, such as urea or guanidinium chloride, seems to be necessary, except when (partially) reduced proteins precipitate. The reduction is carried out best at room temperature at slightly alkaline pH in Tris buffer (0.1 M) or in bicarbonate (0.5 M). Reduction at acid pH can be performed, but is much slower and the resulting thiols are unreactive in respect to alkylation under acidic conditions. All examples given in the chapter are reductions and in situ alkylations. If reductions are to be performed with a very small excess of PBu 3 (5-20%), it is advisable to exclude oxygen completely. This can be achieved as follows: The protein is dissolved in a sealed flask connected with a vacuum pump and a nitrogen cylinder. The flask is evacuated with stirring or shaking and then filled with nitrogen. This procedure is repeated two or three times and reagents are added under positive nitrogen pressure.
232 citations
TL;DR: Staphylococcal protease can be used for the determination of the amino acid sequences of several proteins and proved to be another valuable tool for such studies.
Abstract: Publisher Summary Proteolytic enzymes catalyzing the hydrolysis of peptide bonds involving exclusively the basic amino acid residues lysine and arginine have been available for many years. Trypsin is by far the best known enzyme exhibiting this high degree of specificity and, for that reason, it has played a central role in the studies of the primary structure of proteins. Recently, enzymes that specifically cleave peptide bonds at the carboxyl group of the acidic amino acid residues, aspartic acid and glutamic acid, have been discovered. One of these enzymes, staphylococcal protease, has this specificity and can be further restricted to glutamyl bonds only under certain controlled conditions. This enzyme can be used for the determination of the amino acid sequences of several proteins and proved to be another valuable tool for such studies. Staphylococcal protease shows a marked preference for certain aspartyl bonds when used in ammonium bicarbonate or acetate buffer. The staphylococcal protease is fully active in the presence of 0.2% sodium dodecyl sulfate and retains 50% of its activity in a 4 M urea solution. Digestion under these conditions could be attempted for proteins or peptides that are not readily attacked by the protease under nondenaturing conditions.
228 citations
TL;DR: The properties and merits of the manual mode and its chemical and mechanical implementation are considered and a comparison of the merits of each mode suggests the continued application of manual methods to limited sequencing projects with small budgets.
Abstract: Publisher Summary This chapter considers the properties and merits of the manual mode and its chemical and mechanical implementation. Manual methods have all the options and capabilities of automatic methods plus others that are not yet automated, such as multiple sample processing. Besides being flexible, manual methods are cheap, especially with regard to capital outlay. Any sequencing strategy can be realized in either an automatic or manual mode. A comparison of the merits of each mode suggests the continued application of manual methods to (1) limited sequencing projects with small budgets; (2) large numbers of smaller-sized peptides (to about 20 residues); (3) cases requiring special handling; (4) the characterization of NH 2 -terminal regions for identification; and (5) screening prior to attempts at complete, automatic degradation. Successful sequencing depends more on fundamental understanding of Edman chemistry than on hardware. The factors considered in this chapter are relevant to all approaches, but are most specifically related to liquid-partition with direct identification of the derivatized products.
207 citations
TL;DR: This chapter presents the sequence studies of peptides and proteins using carboxypeptidase Y, an enzyme that removes amino acids as one residue at a time from the carboxyl termini of proteins and peptides.
Abstract: Publisher Summary Carboxypeptidase Y is an enzyme that removes amino acids as one residue at a time from the carboxyl termini of proteins and peptides. This chapter presents the sequence studies of peptides and proteins using carboxypeptidase Y. The assay of peptidase activity is based on the rate of the enzymic hydrolysis of benzyloxycarbonyl-L-phenylalanyl-L-leucine (Z-Phe-Leu). The rate of the reaction can be measured either with the colorimetric ninhydrin method for the estimation of liberated leucine, or spectrophotometrically by the decreases in absorbance at 224 nm. The assay of esterase activity is based on the titrimetric measurement of the release of protons, or upon the spectrophotometric measurement of the change in ultraviolet absorbancy that occurs as a result of the enzymic hydrolysis of Ac-Tyr-OEt. It is found that carboxypeptidase Y hydrolyzes ester and amide substrates of chymotrypsin. The properties of amidase action could be applied to the sequence analyses of peptides with amidated COOH-terminal groups, such as oxytocin and vasopressin.
194 citations
TL;DR: This chapter focuses on sulfhydryl reagents that have been categorized as blocking and labeling groups, reporter groups, cross-linking groups, and affinity labeling groups.
Abstract: Publisher Summary This chapter focuses on sulfhydryl reagents that have been categorized as blocking and labeling groups, reporter groups, cross-linking groups, and affinity labeling groups. Most of these sulfhydryl reagents deliver groups that fall in the category of “blocking and labeling groups,” which may be either reversible or irreversible. These groups are designed to be structurally and chemically relatively innocuous so that when covalently bound, they act merely to block the activity of or to titrate or label (sometimes isotopically) any number of sulfhydryl groups. A second class of sulfhydryl reagents delivers cross-linking groups. They possess one functionality that reacts initially and selectively with sulfhydryls and a second functionality that reacts (often under altered conditions) with another nearby group, which may or may not be another sulfhydryl. For the other two major categories, reporter groups and affinity labeling groups, there is a scarcity of older reagents that may be similarly classified, which react satisfactorily, and are of any general practical utility.
173 citations
TL;DR: Reductive alkylation procedure is applicable to most proteins, except those containing readily reducible components or prosthetic groups, such as pyridoxal phosphate and rhodopsin, or those which are not stable at the required alkaline pH values.
Abstract: Publisher Summary Many simple aldehydes and ketones react rapidly and reversibly with amino groups of proteins. Neither the initial adduct or the Schiff base formed upon dehydration is very stable in dilute aqueous solution, but extensive modification of protein amino groups can be obtained by the reduction of the Schiff base to a stable secondary amine. Reductive alkylation of protein amino groups can, thus, be accomplished with many different aldehydes and ketones under very mild conditions by using sodium borohydride as the reductant. Under the conditions described in the chapter, both α- and ɛ-amino groups are readily modified, but other common protein groups are not affected. Because the modified groups experience only a small change in basicity and, at neutral pH, retain their normal cationic charges, the overall charge and the relative distribution of charged groups in most proteins are not greatly changed by reductive alkylation. By using a large variety of readily available aldehydes and ketones, the size, shape, and hydrophobicity of added substituents can be easily varied. Reductive alkylation procedure is applicable to most proteins, except those containing readily reducible components or prosthetic groups, such as pyridoxal phosphate and rhodopsin, or those which are not stable at the required alkaline pH values.
TL;DR: The recent introduction of fluorescamine, a novel reagent designed after the fluorogenic reaction of ninhydrin with primary amines in the presence of phenylalanine, has now provided a strong alternative to ninHydrin for detection of peptides.
Abstract: Publisher Summary The recent introduction of fluorescamine, a novel reagent designed after the fluorogenic reaction of ninhydrin with primary amines in the presence of phenylalanine, has now provided a strong alternative to ninhydrin for detection of peptides Fluorescamine reacts with primary amino groups of peptides almost instantaneously at room temperature in aqueous solution at pH 75-9, to form a fuorescent compound The reagent is subsequently hydrolyzed to yield a watersoluble, nonfluorescent product Fluorescamine itself is not fluorescent Ammonia in solution yields little or no fluorescence with fluorescamine These features and the great sensitivity attainable in the fluorescence measurement have made fiuorescamine increasingly popular in amino acid and peptide analysis Another development in recent years is the rediscovery of o -phthalaldehyde as a general reagent for amino acids and peptides when used in the presence of 2-mercaptoethanol This reagent has recently been applied to the microanalysis of amino acids and peptides
TL;DR: High-performance liquid chromatography of amino acid derivatives, although developed primarily for monitoring amino acid sequencing, offers a sensitive method for amino acid analysis from protein and peptide hydrolysates.
Abstract: Publisher Summary The detection and quantitative analysis of amino acids in subnanomole quantities can be accomplished by several methods. Thin-layer chromatography (TLC) has been used to detect PTH-derivatives at subnanomole levels. Combined with autoradiography, TLC techniques have been described for subpicomole levels of tritiated dansyl amino acids. Gas chromatography of amino acid derivatives has been used to analyze amino acids in picomole range. High-performance liquid chromatography of amino acid derivatives, although developed primarily for monitoring amino acid sequencing, offers a sensitive method for amino acid analysis from protein and peptide hydrolysates. When amino acid derivatives must be synthesized prior to analysis, it is necessary to ensure quantitative and reproducible derivative yields, as well as derivative stability. By carrying reagent blanks and low-level standard amino acid mixtures through the hydrolysis procedures, it is possible to correct the contamination effects and losses during hydrolysis.
TL;DR: Thin layer, two-dimensional techniques are based on the same fundamental combination of chromatographic and electrophoretic separations originally utilized on large filter papers, but are carried out on much smaller sheets coated with thin layers of cellulose or silica gel.
Abstract: Publisher Summary Thin layer, two-dimensional techniques are based on the same fundamental combination of chromatographic and electrophoretic separations originally utilized on large filter papers, but are carried out on much smaller sheets coated with thin layers of cellulose or silica gel Whereas standard, two-dimensional separations require 1-5 mg of digested protein, peptide maps on thin layers can easily be obtained with 10-50 μg (ie, 02-10 nmol) of material Moreover, by using radioisotope-labeled peptides, the level of sensitivity can be extended even further In addition to the sensitivity and speed of separation, thin-layer methods do not require specialized high voltage supplies or electrophoretic chambers that are capable of dissipating large amounts of heat There are additional advantages of thin-layer methods in particular situations For example, in the comparison of normal and genetically or chemically modified proteins, it is a distinct advantage to utilize a system in which both the normal and modified protein digests can be simultaneously subjected to electrophoresis or chromatography, thereby assuring optimal comparisons
TL;DR: Thermolysin has been used to probe the three-dimensional structure of native proteins, but in most of these cases, the sequences of the protein substrates were known, and the enzyme does seem to cleave native proteins to a greater extent than most proteases.
Abstract: Publisher Summary Thermolysin is a thermostable, extracellular metalloendopeptidase isolated from the culture filtrates of the thermophilic organism, Bacillus thermoproteolyticus. Thermolysin can be applied as a tool in structural analysis both in the elucidation of protein covalent structures and in defining certain elements of the tertiary structures of proteins. Because of its broad specificity, thermolysin is usually employed in the secondary cleavage of tryptic and chymotryptic peptides during the course of the sequence analysis of the protein of interest. The use of thermolysin in the cleavage of peptides that have already undergone prior hydrolysis with trypsin and chymotrypsin, in effect, increases its specificity. Thermolysin has been used to probe the three-dimensional structure of native proteins, but in most of these cases, the sequences of the protein substrates were known. Two properties of thermolysin are important in regard to its potential application in the generation of large polypeptide fragments from a native protein. First, the enzyme does seem to cleave native proteins to a greater extent than most proteases. The second property of thermolysin important in this regard is its thermostability.
TL;DR: Oxidative conversion of methionine to its sulfoxide has been utilized in peptide synthesis as a means of avoiding side reactions that might otherwise occur during chemical manipulations in the synthesis of a polypeptide.
Abstract: Publisher Summary The conversion of methionine to the corresponding sulfoxide residue in proteins has been used to study the influence of the former on the physicochemical and biological properties of proteins. The availability of procedures for regenerating methionine from the sulfoxide permits the verification of the specificity of the conversion and also allows reversible protection of the thioether group during chemical modification of other amino acid residues. Because of the nucleophilic character of the thioether function, methionine is reactive toward the majority of electrophilic reagents used for chemically modifying amino acid side chains in proteins. Oxidative conversion of methionine to its sulfoxide has been utilized in peptide synthesis as a means of avoiding side reactions that might otherwise occur during chemical manipulations in the synthesis of a polypeptide. In addition, methionine oxidation is a potentially useful technique in the preparation of overlapping peptides for the purpose of sequence analysis because methionine sulfoxide peptide bonds are not cleaved in the cyanogen bromide reaction.
TL;DR: The use of disulfides formed from the 6-thio analog of ATP followed by cyanide, or direct cyanylation of protein SH groups with a6-thiocyano analogue of ATP has led to the incorporation of label and specific clearage in several cases.
Abstract: Publisher Summary The SH groups of denatured proteins can be converted quantitatively to SCN groups with 2-nitro-5-thioeyanobenzoate (NTCB) 1,2, or with other reagents. NTCB is readily synthesized and can be labeled with [ 14 C] cyanide. Exposure to pH 8-9 results in cleavage at the amino group of the modified cysteine residue in excellent yield. Because disulfides do not react with NTCB, selective cleavage at cysteine can be obtained in the presence of cystine on the basis of this reaction. Specific SH groups of undenatured papain, aspartate transcarbamylase, isocitrate dehydrogenase, aspartate aminotransferase, NAD-specific glutamate dehydrogenase, glyceraldehyde-3-P dehydrogenase, and myosin can be modified with NTCB, or with 5,5'-dithiobis plus cyanide. Specific cleavage at cysteine residues specifically modified in native proteins provides a convenient method for locating residues. The use of disulfides formed from the 6-thio analog of ATP followed by cyanide, or direct cyanylation of protein SH groups with a 6-thiocyano analog of ATP has led to the incorporation of label and specific clearage in several cases.
TL;DR: This chapter describes methods involving the potentially irreversible glutamine antagonists which have proved to be versatile in studies on glutamine utilization by enzymes.
Abstract: Publisher Summary Glutamine plays a central role in the nitrogen metabolism of both prokaryotic and eukaryotic organisms. There are several compounds with structural similarity to glutamine but containing different functional groups in place of the normal side-chain amide. Some of these compounds might be expected to interfere with the utilization of glutamine only by inhibition in a competitive and reversible manner whereas others contain an alkylating group, such as a diazoketone (I,IV), ehloroketone (III), diazoaeetyl ester (II), or chloroisoxazole (V), which confers the potential of reacting with an amino acid in or near the glutamine binding site. Such reaction would result in covalent attachment of the inhibitor and, hence, irreversible inhibition. This chapter describes methods involving the potentially irreversible glutamine antagonists which have proved to be versatile in studies on glutamine utilization by enzymes. It explains the preparation and properties of glutamine analogs and use of glutamine analogs.
TL;DR: The purpose of this chapter is to present a descriptive overview of the current status of affinity labeling; the evolution of the concepts and techniques, the most general picture of the principles involved, the terminology and the criteria commonly applied, and the current and prospective applications of affinitylabel techniques.
Abstract: Publisher Summary Affinity labeling by definition applies to all molecules possessing a site that binds another molecule with a certain degree of specificity and affinity In biology the proteins are the obvious ligand-binding molecules, and in practice the affinity labeling techniques have primarily involved proteins The complete understanding of how proteins carry out their biological function requires a detailed understanding of how this binding site is constituted, and this has become the key problem in the area of protein structure-function analysis The purpose of this chapter is to present a descriptive overview of the current status of affinity labeling; the evolution of the concepts and techniques, the most general picture of the principles involved, the terminology and the criteria commonly applied, and the current and prospective applications of affinity labeling techniques In this chapter the discussion is kept general and is based on hypothetical examples The chapter explains the principles and types of reagents, the Covalent Bond formation, interpretations, variations and special considerations and the use of affinity labeling
TL;DR: 3'- and 5'-FSBA may be useful in the affinity labeling of receptor proteins as well as of the regulatory and active sites of enzymes within the constraints imposed by the limitations of solubility and stability of these compounds.
Abstract: Publisher Summary Among the coenzymes and regulatory compounds involved in biochemical reactions, adenine nucleotides are present in a large proportion of cases. Adenosine triphosphate and the corresponding diphosphate are required for many kinase reactions, in which a phosphoryl group is transferred to a variety of substrates; and adenosine is part of the coenzymes, DPN and TPN, that participate in most dehydrogenase reactions. In addition, these compounds are important in activating or inhibiting the activity of a number of regulatory enzymes. This chapter also explains preparation of adenosine analogs, and examples of reactions of 3'-FSBA and 5'-FSBA with particular proteins. It concludes that 3'- and 5'-FSBA may be useful in the affinity labeling of receptor proteins as well as of the regulatory and active sites of enzymes. Within the constraints imposed by the limitations of solubility and stability of these compounds, the fluorosulfonylbenzoyladenosines thus may have broad applicability in the labeling of adenine nucleotide sites in proteins.
TL;DR: In this paper, the authors used column packings, packing techniques, column designs and high-pressure, and pulseless pumps for the analysis of phenylthiohydantoins and 1-dimethylaminonaphthalene-5-sulfonyl (dansyl) amino acids.
Abstract: Publisher Summary Recently, liquid chromatographic analyses have been performed with the speed and resolving power characteristic of gas chromatography. Improved column packings, packing techniques, column designs and high-pressure, and pulseless pumps are largely responsible for the superior performance of gas chromatography. High-performance liquid chromatography features low dead volume, narrow-bore columns, and small, uniform packings. High-performance separations have been achieved by each of the four major separation mechanisms: adsorption, molecular exclusion, ion-exchange, and partition. Changes in temperature and flow rate can markedly influence column performance. Recently, Zorbax-ODS columns have been found giving higher back pressures. The inlet pressure must be 500-1000 psi to obtain a flow rate of 0.8 ml/min. Many amino acid derivatives may be easily separated by partition chromatography and determined with high sensitivity using ultra-violet (UV) or fluorescence detectors. Silica and reversed phase columns have been especially useful for the analysis of phenylthiohydantoins and of 2,4-dinitrophenyl and 1-dimethylaminonaphthalene-5-sulfonyl (dansyl) amino acids.
TL;DR: This chapter discusses that the two general approaches are available for the synthesis of aza-peptides and aza -amino acid derivatives.
Abstract: Publisher Summary Aza-amino acid residues are analogs of amino acids in which the α-CH has been replaced by a nitrogen atom. This substitution has a profound effect on the reactivity of aza-peptides, i.e., those containing aza-amino acid residues. In particular, it has been shown that azapeptide p -nitrophenyl esters are inhibitors and active site titrants of several serine proteases. Inhibition of these enzymes is believed to arise from the acylation of the active-site serine residue yielding an acylated enzyme, which is substantially less reactive toward deacylation than a normal acylated enzyme owing to the influence of the adjacent nitrogen atom. At present, there is no evidence that the active site serine residue is actually the site of reaction. However, the close structural resemblance of aza-peptides to normal peptides substrates of serine proteases points strongly to the serine residue as the one being labeled. This chapter discusses that the two general approaches are available for the synthesis of aza-peptides and aza-amino acid derivatives. The first method involves the reaction of an acyl hydrazide with an aldehyde followed by reduction with sodium borohydride and reaction with p -nitrophenyl chloroformate. The second method approach involves the reaction of a monosubstituted hydrazine with an ester.
TL;DR: This chapter concludes that mechanism-based irreversible enzyme inhibitors are more than an enzymological curiosity and can be useful as general selective irreversible enzymes inhibitors.
Abstract: Publisher Summary There are two extreme forms of mechanism for specific irreversible enzyme inhibitors. One comprises the classical affinity labeling agents, which are substrate analogs containing chemically reactive functional groups. The specific binding of these inhibitors to the active-site regions of the enzyme ensures a greater probability of chemical reaction with a residue within this region as opposed to nonspecific chemical reactions. This chapter explains the irreversible enzyme inhibitors. It defines the conditions on which the successful design of mechanism-based irreversible enzyme inhibition is dependent. It cites specific reagents like β-γ – Unsaturated Molecules, diazoesters and ketones, Allyl chloride and sulfates, vinyl chlorides and other reagents. It also explains the synthsis of Vinyl Glycine and p-Nitrophenyl Diazoacetate. The chapter concludes that mechanism-based irreversible enzyme inhibitors are more than an enzymological curiosity and can be useful as general selective irreversible enzyme inhibitors.
TL;DR: Clostripain is a sulfhydryl protease with a narrow specificity range limited to the carboxyl peptide linkage of positively charged amino acids that possesses both esterase-amidase, as well as protease activities.
Abstract: Publisher Summary Clostripain is a sulfhydryl protease with a narrow specificity range limited to the carboxyl peptide linkage of positively charged amino acids. This chapter presents the enzymic properties of clostripain relevant to its specificity and potential utility. Clostripain markedly prefers arginine over lysine residues. Thus, under the conditions of controlled hydrolysis, peptide bond cleavage can be limited to arginine sites, including trypsin-resistant arginylprolyl bonds. Clostripain also possesses both esterase-amidase, as well as protease activities. Clostripain offers substantial advantages to the investigators of primary sequence. An example of a sequence problem in which clostripain was used to solve an overlap is provided by structural studies on a lysozyme produced by a species of Chaloropsis. Clostripain has been also used as an aid in the primary structure determination of tuftsin and as a tool in structure-function studies on glucagon.
TL;DR: This chapter discusses the synthesis, kinetics of reaction and applications of the reaction of serine proteases with halomethyl ketones, one of the classic demonstrations of the value of affinity labels for enzyme studies.
Abstract: Publisher Summary The use of the chloromethyl ketones Tos-PheCH 2 C1 (TPCK) and Tos-LysCH 2 C1 (TLCK) as specific reagents for chymotrypsin and trypsin, respectively, is one of the classic demonstrations of the value of affinity labels for enzyme studies. Subsequently, halomethyl ketones were used for characterization of functional groups and sequences in active sites, as probes of structure by providing labeled proteins that contain spectroscopic handles, in crystallographic studies of substrate binding sites, and in the study of the biological function of proteases. The reaction of a serine protease with most substrate-related haloketones probably first involves the formation of an enzyme inhibitor complex in which the inhibitor is recognized by specific interactions between the side chain of the P 1 amino acid residue and the S 1 or primary substrate binding site of the enzyme. Irreversible inhibition takes place within this complex by covalent bond formation between the active-site histidine residue and a methylene group of the inhibitor. This chapter discusses the synthesis, kinetics of reaction and applications of the reaction of serine proteases with halomethyl ketones.
TL;DR: The modification of the earlier synthesis of 8-N3-cAMP is detailed, which is easier to perform and consistently gives yields of 60–80 per cent without dilution of the specific radioactivity.
Abstract: Publisher Summary The use of photoaffinity reagents to investigate the binding of ligands to macromolecules is a relatively new technique and is being rapidly developed. This technique has been successfully used to identify ATP and cAMP binding proteins of cells, cell membranes, and some purified protein kinases. The reagent successfully used to photolabel cAMP binding sites was 8-azidoadenosine 3',5'-cyclic monophosphate (8-N3-cAMP). In the absence of activating light, 8-N-cAMP is a good biological substitute for cAMP. For 8-N3-cAMP to be of optimal value it must be synthesized in radioactive form without loss of the high specific activity of the starting compound. This chapter details the modification of the earlier synthesis of 8-N3-cAMP, which is easier to perform and consistently gives yields of 60–80 per cent without dilution of the specific radioactivity. This procedure has also been used to prepare P- and C-labeled 8-azidoadenosine monophosphate. It cannot be used on compounds containing tritium-labeled adenosine without loss of the 3H on the 8 position.
TL;DR: A significant step in this direction could be made by finding a reagent that activates carboxyl groups without side reactions, as the greatest need in solid-phase sequencing is a completely general and reliable means of immobilizing peptides.
Abstract: Publisher Summary The technique of the solid-phase Edman degradation relies on efficient procedures for attaching peptides or proteins to amino-substituted insoluble supports. Because sequencing proceeds from the NH2 terminus, it is desirable to attach peptides at the COOH terminus. If the activation of COOH-terminal carboxyl is used for coupling, the problem then arises of selectively activating the COOH-terminal group without simultaneously activating the side chains of aspartic and glutamic acids. Considerable effort has gone into the search for a completely general method; however, none has yet been found. Instead, several methods selective for certain types of peptides are devised to negotiate with this problem, including NH2-terminal blocking, carbonyldiimidazole activation, carbodiimide activation, and attachment at homoserine, along with combined attachment methods. Still the greatest need in solid-phase sequencing is a completely general and reliable means of immobilizing peptides. A significant step in this direction could be made by finding a reagent that activates carboxyl groups without side reactions.
TL;DR: 2,4,6-Tribromo-4-methylcyclohexadienone is found to be less reactive than NBS, giving similar yields of cleavage, but extensive modification of tyrosine, histidine, and sulfur amino acids is observed in this case.
Abstract: Publisher Summary Procedures for effecting specific cleavage of tryptophanyl peptide bonds of proteins have been intensively sought in the past decade. The reagents tested in such procedures include periodate, ozone, and in particular brominating agents. N-Bromosuccinimide (NBS) was the first reagent proposed for the cleavage of tryptophanyl peptide bonds, but various side reactions of this extremely reactive reagent with other amino acid side chains, as well as poor yields in peptide bond fission, hampered practical applications of the reagent. 2,4,6-Tribromo-4-methylcyclohexadienone is found to be less reactive than NBS, giving similar yields of cleavage, but extensive modification of tyrosine, histidine, and sulfur amino acids is observed in this case. Positive halogen reagents, such as ICl, iodide oxidized with Chloramine-T, N-iodosuccinimide, and active iodine generated with H 2 O 2 , iodide, and a peroxidase, also give moderate yields of cleavage. A mixture of dimethyl sulfoxide (DMSO) and concentrated aqueous HBr has been recently used as a brominating agent to effect selective cleavage of tryptophanyl peptide bonds in peptides and proteins.
TL;DR: The successful chemical coupling of arylazido groupings to adenine nucleotide and the extension of this methodology to the pyridine nucleotides is described.
Abstract: Publisher Summary The ATP molecule contains three distinct structural groupings: the adenine base, the ribose sugar, and the triphosphate component. On the basis of the interaction of actomyosin with ATP analogs modified in these positions, a model of the enzyme substrate complex has been postulated. The nucleotide binding is thought to involve an NH group at the trinitrobenzene sulfonate interacting site as well as conjugation of triphosphate with asparagine via metal interaction and binding of the terminal pyrophosphate to a sulfhydryl and guanidinium group at the active site. An important aspect of these actomyosin–nucleotide analog studies is the appreciation that the ribose ring can be drastically modified without affecting markedly the rate of myosin-catalyzed hydrolysis of the analog. This chapter describes the successful chemical coupling of arylazido groupings to adenine nucleotides and the extension of this methodology to the pyridine nucleotides. The synthesis of arylazido analogs of CoA and tetrodotoxin are mentioned.
TL;DR: This chapter examines the synthetic procedures of carboxypeptidases and the active site of carboxespecific exopeptidase A and B, and implies that a reagent designed for reaction with carboxypesptid enzyme A may also be effective withcarboxypePTidase B.
Abstract: Publisher Summary Carboxypeptidases A (EC 3.4.12.2) and B (EC 3.4.12.3) are pancreatic exopeptidases. carboxypeptidase A preferentially hydrolyzes terminal peptide bonds in which the amino donor is a hydrophobic amino acid whereas the specificity of carboxypeptidase B is directed toward the terminal basic amino acids. The design of reagents for affinity labeling of these enzymes is based on their specificity, that is, a free a-carboxylate group, a hydrophobic or basic side chain, and a reactive group. However, it must be kept in mind that carboxypeptidase B has, in addition to its known specificity toward basic substrates, an intrinsic activity with specificity similar to that of carboxypeptidase A. This implies, in general, that a reagent designed for reaction with carboxypeptidase A may also be effective with carboxypeptidase B, whereas a nonbasic reagent used for carboxypeptidase B may also act with carboxypeptidase A. This chapter examines the synthetic procedures of carboxypeptidases and the active site of carboxypeptidases,