SH Group Of Cysteine Research Articles

Mechanisms of Carboxymethylation of Bovine Pancreatic Nucleases by Haloacetates and Tosylglycolate

Abstract Certain histidine residues in bovine pancreatic ribonuclease and deoxyribonuclease react with iodoacetate about 1000 times faster than free histidine reacts. The mechanisms of these facilitated, active-site-directed reactions were investigated by study of the kinetics of carboxymethylation with reagents with varied leaving groups: chloride, bromide, iodide, and tosylate. With ribonuclease, the rate of reaction gave hyperbolic dependence on the concentration of reagent. The data fit a Michaelis-Menten mechanism, with dissociation constants of 8 to 24 mm for the various reagents. The similar magnitudes of the constants lead to the conclusion that the reagents bind (reversibly) in a common way to the enzyme. The pseudo bimolecular rate constants for reaction (after correction for the differing inherent reactivities of the reagents) were also similar, indicating that the leaving group is not very important in the mechanism. These studies support the proposal that the reaction of haloacetates with ribonuclease is facilitated by ionic attraction of the carboxylate ion by a protonated imidazole while an unprotonated imidazole displaces the halide ion. In contrast, the kinetics of carboxymethylation of the Cu2+-Tris complex of deoxyribonuclease showed that the reagents bound loosely, with dissociation constants of 130 mm or larger. Furthermore, the corrected, pseudo bimolecular rate constants increased as the size of the leaving group decreased. The larger groups may be more sterically hindered in the reaction; alternatively, the smaller groups may be attracted more to the electrophilic Cu2+. In either case, during carboxymethylation, both the carboxylate ion and the leaving group interact with DNase. While the carboxymethylation of RNase may be facilitated by the precise orientation of the reagent, the carboxymethylation of DNase may be facilitated by electrophilic catalysis. Tosylglycolate (carboxymethyl p-toluenesulfonate) was prepared and tested as an analog of the haloacetates. The reagent was about one-eighth as reactive as iodoacetate with a pyridine base and reacted with the —SH group of cysteine. It carboxymethylated histidines 119 and 12 of ribonuclease with more preference for histidine 119 than did iodoacetate.

Stability of nitrofuran derivatives to cysteine, gastro-intestinal contents and light

Stability of a series of nitrofuran derivatives against cysteine, content of digestive tract, and light was examined. 1) (a) Nitrofuran derivatives are decomposed by the SH group of cysteine (H3N+·R·S-). There is a certain relationship between the chemical structure and stability against cysteine (Chart 1).[chemical formula] (b) Stability of the derivatives with vinylog structure can be improved to some extent by N-oxygenation of the hetrocyclic nitrogen. (c) There is a positive correlation between stability to cysteine and therapeutic effect of typhoid-infected mice by oral administration in derivatives with vinylog structure. 2) Nitrofuran derivatives are decomposed by the content in the digestive tract lower than the small intestine and this decomposition is especially marked by cecal content. This decomposition is due to insoluble protein, enzyme, or bacterial SH in the content of the digestive tract. 3)(a) Nitrofuran derivatives in aqueous solution are decomposed by sunlight. Relationship between chemical structure and stability to light is approximately similar to that in the case of cysteine but not completely identical. (b) Stability of the aqueous solution to light depends on the concentration of derivatives, stability being better the higher the concentration. (c) Stability of the derivatives in various solvents became better with higher dielectric constant of the solvent. 4) Decomposition products of nitrofuran derivatives have no antibacterial activity.

Rate assay for estimation of thiol affinity of sulfhydryl-reactive agents: Estimation of SH-reactivity

An interaction between cysteine SH groups and β- nitrostyrene was suggested from alterations in the ultraviolet spectrum of β-nitrostyrene following the addition of cysteine, but not serine. In the presence of β-nitrostyrene or phenylmercuric chloride, the rate of color development resulting from the reaction of cysteine and DTNB was reduced; increasing the concentration of β-nitro- styrene resulted in increased interference in the color development. Excess DTNB did not mitigate the influence of β-nitrostyrene on color development; however, excess cysteine was found to overcome the effects of the β-nitrostyrene on the development of color. The implication of this interference on single point SH content determinations is discussed. Application of inhibition of the cysteine DTNB reaction to estimate SH-reactivity of thiol-reactive agents is described.

The chemical reactions of the haemagglutinins and neuraminidases of different strains of influenza viruses. I. Effect of reagents reacting with amino acids in the active centres.

SUMMARYStudies of the chemical reactions of the haemagglutinins and neuraminidases of eight strains of influenza viruses have been made by the use of chemical reagents reacting with chemically active groups in the protein molecule. The results indicate a close resemblance between the active centres of the haemagglutinins and neuraminidases in all the strains tested. In all cases the activities were unaffected by reagents reacting with the —SH group of cysteine, the —CH3S group of methionine, the amino group of lysine, the guanidyl group of arginine, or the indole ring of tryptophan. In all cases both the haemagglutinating and enzymic activities were reduced or destroyed by agents reacting with amide groups or reacting with both tyrosine and histidine.By the use of iodine under conditions in which tyrosine reacts but not histidine, and fluorodinitrobenzene under conditions in which histidine reacts more strongly than tyrosine, it was possible to detect a number of different active centres.(1) An active centre containing histidine and an amide group but not containing tyrosine was present in all the virus strains and was the only centre detectable in A1 and A2 strains. This type of centre appeared to possess both haemagglutinating and neuraminidase activity.(2) Active centres containing tyrosine and an amide group were detected in strains of A and B viruses. There was some evidence suggesting that tyrosine-containing centres were of two types: one possessing both haemagglutinating and enzymic activity while the other was a haemagglutinin without neuraminidase activity.The results could be explained by supposing that the presence of histidine in the active centre was essential for neuraminidase activity and that enzymically active tyrosine-containing centres also contained histidine, but that tyrosine could substitute for histidine, but that tyrosine could substitute for histidine in haemagglutinating centres.

Direct spectrophotometric estimation of ferrocyanide and its possible uses in sulfhydril oxidation studies

1. 1. The direct spectrophotometry of ferrocyanide in the presence of ferricyanide has been examined. 2. 2. Ferrocyanide at a concentration of 1 × 10 −5 M is easily measurable in the presence of a 50-fold excess of ferricaynide. 3. 3. The measurement may be performed over a wide range of conditions, including those suited to the oxidation of most biological SH groups. 4. 4. The method has been compared with the Prussian blue method for the estimation of oxidation of the SH group of cysteine. 5. 5. The method has many advantages over the Prussian blue method, including its possible use in direct kinetic measurements.

Failure of N-ethyl maleimide to react with sulfhydryl groups of intact human erythrocytes.

Certain sulfhydryl reagents such as p-chloromercuribenzoic acid hemolyze human erythrocytes in a predictable manner. N-ethyl maleimide does not hemolyze human erythrocytes although it reacts freely with the –SH groups of cysteine, glutathione and ergothionine. Also N-ethyl maleimide reacts avidly with the –SH groups of human erythrocytes previously hemolyzed by freezing. These findings indicate the envelope of the intact erythrocyte prevents access of N-ethyl maleimide to the critical –SH groups of the intact erythrocyte or that freezing exposes the crucial –SH groups by altering the natural configuration of the proteins. Submitted on April 6, 1956

SOME FACTORS WHICH INFLUENCE THE OXIDATION OF SULFHYDRYL GROUPS

1. Cyanide inhibits the oxidation of the SH groups of cysteine and denatured egg albumin by the uric acid reagent. 2. At pH 4.8 cysteine is oxidized by the uric acid reagent and by ferricyanide in the presence but not in the absence of added copper sulfate. 3. In neutral solution, the uric acid reagent oxidizes the SH groups of denatured egg albumin in the presence of urea but not in the presence of alkyl sulfate or in the absence of denaturing agents. 4. Ferricyanide oxidizes the SH groups of neutral denatured egg albumin even in the presence of alkyl sulfate or, if precautions are taken to avoid aggregation, in the absence of denaturing agents. 5. In acid solution, ferricyanide does not oxidize the SH groups of denatured egg albumin completely. The oxidation is more complete, however, in the presence of urea than in the presence of alkyl sulfate, and more complete in the presence of guanidine hydrochloride than in the presence of urea. 6. The uric acid reagent which does not oxidize the SH groups of neutral denatured but unhydrolyzed egg albumin in the absence of denaturing agents does, under the same conditions, oxidize the SH groups of egg albumin partially hydrolyzed by pepsin. 7. At pH 4.8 in alkyl sulfate solution ferricyanide oxidizes the SH groups of digested egg albumin more completely than the SH groups of denatured but undigested egg albumin.

THE REACTIONS OF DENATURED EGG ALBUMIN WITH FERRICYANIDE

The following facts have been established experimentally. 1. In the presence of the synthetic detergent, Duponol PC, there is a definite reaction between dilute ferricyanide and denatured egg albumin. 0.001 mM of ferrocyanide is formed by the oxidation of 10 mg. of denatured egg albumin despite considerable variation in the time, temperature, and pH of the reaction and in the concentration of ferricyanide. 2. If the concentration of ferricyanide is sufficiently high, then the reaction between ferricyanide and denatured egg albumin in Duponol solution is indefinite. More ferrocyanide is formed the longer the time of reaction, the higher the temperature, the more alkaline the solution, and the higher the concentration of ferricyanide. 3. Denatured egg albumin which has been treated with formaldehyde or iodoacetamide, both of which abolish the SH groups of cysteine, does not reduce dilute ferricyanide in Duponol PC solution. 4. Cysteine is the only amino acid which is known to have a definite reaction with ferricyanide or which is known to react with dilute ferricyanide at all. The cysteine-free proteins which have been tried do not reduce dilute ferricyanide in Duponol PC solution. 5. Concentrated ferricyanide oxidizes cystine, tyrosine, and tryptophane and proteins which contain these amino acids but not cysteine. The reactions are indefinite, more ferrocyanide being formed, the higher the temperature and the concentration of ferricyanide. 6. The amount of ferrocyanide formed from denatured egg albumin and a given amount of ferricyanide is less in the absence than in the presence of Duponol PC. 7. The amount of ferrocyanide formed when denatured egg albumin reacts with ferricyanide in the absence of Duponol PC depends on the temperature and ferricyanide concentration throughout the whole range of ferricyanide concentrations, even in the low range of ferricyanide concentrations in which ferricyanide does not react with amino adds other than cysteine. The foregoing results have led to the following conclusions which, however, have not been definitely proven. 1. The definite reaction between denatured egg albumin in Duponol PC solution and dilute ferricyanide is a reaction with SH groups whereas the indefinite reactions with concentrated ferricyanide involve other groups. 2. The SH groups of denatured egg albumin in the absence of Duponol PC react with iodoacetamide and concentrated ferricyanide but they do not all react rapidly with dilute ferricyanide. 3. Duponol PC lowers the ferricyanide concentration at which the SH groups of denatured egg albumin react with ferricyanide. The SH groups of denatured egg albumin, however, are free and accessible even in the absence of Duponol PC.