Disulfide bond modification

Figure 17. The effect of disulfide bond modification of turkey ovomucoid by alkali on inhibitory activity against trypsin (T), a-chymotrypsin, (C), and subtilisin (S). Turkey ovomucoid (O.lOmM) was treated with alkali (lOOmM NaOH) at 23°C. Sulfhydryl content (moles per mole of protein)...

Friedman, M., Grosjean, O.K. and Zahnley, J.C. (1982c). Effect of disulfide bond modification on the structure and activities of enzyme inhibitors. "Mechanisms of Food Protein Deterioration", J.P. Cherry, Ed., ACS Symp. Series,... 

FIGURE 5.18 Methods for cleavage of disulfide bonds in proteins, (a) Oxidative cleavage by reaction with performic acid, (b) Reductive cleavage with snlfliydryl compounds. Disulfide bridges can be broken by reduction of the S—S link with snlfliydryl agents such as 2-mercaptoethanol or dithiothreitol. Because reaction between the newly reduced —SH groups to re-establish disulfide bonds is a likelihood, S—S reduction must be followed by —SH modification (1) alkylation with iodoac-etate (ICH,COOH) or (2) modification with 3-bromopropylamine (Br— (CH,)3—NH,). 

DNA sequencing reveals the order in which amino acids are added to the nascent polypeptide chain as it is synthesized on the ribosomes. However, it provides no information about posttranslational modifications such as proteolytic processing, methylation, glycosylation, phosphorylation, hydroxylation of prohne and lysine, and disulfide bond formation that accompany mamra-tion. While Edman sequencing can detect the presence of most posttranslational events, technical hmitations often prevent identification of a specific modification. 

Neurotoxins present in sea snake venoms are summarized. All sea snake venoms are extremely toxic, with low LD5Q values. Most sea snake neurotoxins consist of only 60-62 amino acid residues with 4 disulOde bonds, while some consist of 70 amino acids with 5 disulfide bonds. The origin of toxicity is due to the attachment of 2 neurotoxin molecules to 2 a subunits of an acetylcholine receptor that is composed of a2 6 subunits. The complete structure of several of the sea snake neurotoxins have been worked out. Through chemical modification studies the invariant tryptophan and tyrosine residues of post-synaptic neurotoxins were shown to be of a critical nature to the toxicity function of the molecule. Lysine and arginine are also believed to be important. Other marine vertebrate venoms are not well known. 

Ethylenimine may be used to introduce additional sites of tryptic cleavage for protein structural studies. In this case, complete sulfhydryl modification is usually desired. Proteins are treated with ethylenimine under denaturing conditions (6-8 M guanidine hydrochloride) in the presence of a disulfide reductant to reduce any disulfide bonds before modification. Ethylenimine may be added directly to the reducing solution in excess (similar to the procedure for Aminoethyl-8 described previously) to totally modify the —SH groups formed. 

The 4,4 -dipyridyl disulfide can be used in aqueous solutions, but it has been found that modification of proteins with this reagent yields rapid disulfide bond formation. Only when 2-iminothiolane is used in tandem with 4,4 -dipyridyl disulfide can 4-dithiopyridyl groups be introduced into proteins (King et al., 1978) (Section 4.1, this chapter). This is due to disulfide interchange reactions predominating without the addition of 2-iminothiolane. 

Figure 21.5 SPDP can be used to modify both an antibody and a toxin molecule for conjugation purposes. In this case, the antibody is thiolated to contain a sulfhydryl group by modification with SPDP followed by reduction with DTT. A toxin molecule is then activated with SPDP and reacted with the thiolated antibody to effect the final conjugate through a disulfide bond.

Chemical changes include posttranslational modifications including glycosy-lation, phosphorylation, disulfide bond formation and exchange (scrambling), proteolysis or hydrolysis, and deamidation or oxidation of amino acids.4... 

Cysteine disulfide formation is one of the most important posttranslational modifications involved in protein structure. Disulfides play a crucial role in maintaining the structure of many proteins including insulin, keratin, and many other structurally important proteins. While the cytoplasm and nucleus are reducing microenvironments, the Golgi and other organelles can have oxidizing environments and process proteins to contain disulfide bonds (Scheme 5). 

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