The study describes methods for the preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. The reduction of disulfide bonds in proteins is essential for structural analysis. Tryptophan-free proteins like insulin and ribonuclease can be oxidized with performic acid, but proteins containing tryptophan produce undesirable side products. To avoid this, reduction with mercaptoethanol in 8 M urea and subsequent alkylation with iodoacetate is preferred. This method effectively reduces disulfide bonds and alkylates sulfhydryl groups, minimizing side reactions with methionine, lysine, histidine, and tyrosine residues. The study also improves hydrolysis conditions for carboxymethylated proteins, allowing precise evaluation of alkylation efficiency. Gel filtration on Sephadex G-75 is used to detect small fragments formed during reduction. The preparation of reduced and carboxymethylated proteins at a 50-mg scale involves reducing proteins with mercaptoethanol, alkylating with iodoacetate, and purifying via Sephadex G-75. For larger-scale preparations, a desalting procedure with IRC-50 resin is used. Sodium borohydride can also be used for reduction, but it may cause side reactions. Alkylation with iodoacetate is effective in minimizing these side reactions. Amino acid analyses confirm the success of the procedures, showing quantitative yields of S-carboxymethylcysteine. The study also demonstrates the use of the reduced and carboxymethylated phenylalanyl chain of insulin as a substrate to test the purity of trypsin. Chromatographic separation of peptides from enzymatic hydrolysis is performed using a 15-cm column of the amino acid analyzer. The results show that trypsin hydrolyzes arginyl bonds more slowly when X and Y carry negatively charged groups. The presence of cysteic acid residues reduces the susceptibility to trypsin action compared to S-carboxymethylcysteine residues. The study concludes that reduced and carboxymethylated proteins can be used for structural studies, provided that thioether sulfur of methionine and indole group of tryptophan are protected from oxidation during peptide separation. The methods described provide a reliable way to prepare and analyze proteins for structural studies.The study describes methods for the preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. The reduction of disulfide bonds in proteins is essential for structural analysis. Tryptophan-free proteins like insulin and ribonuclease can be oxidized with performic acid, but proteins containing tryptophan produce undesirable side products. To avoid this, reduction with mercaptoethanol in 8 M urea and subsequent alkylation with iodoacetate is preferred. This method effectively reduces disulfide bonds and alkylates sulfhydryl groups, minimizing side reactions with methionine, lysine, histidine, and tyrosine residues. The study also improves hydrolysis conditions for carboxymethylated proteins, allowing precise evaluation of alkylation efficiency. Gel filtration on Sephadex G-75 is used to detect small fragments formed during reduction. The preparation of reduced and carboxymethylated proteins at a 50-mg scale involves reducing proteins with mercaptoethanol, alkylating with iodoacetate, and purifying via Sephadex G-75. For larger-scale preparations, a desalting procedure with IRC-50 resin is used. Sodium borohydride can also be used for reduction, but it may cause side reactions. Alkylation with iodoacetate is effective in minimizing these side reactions. Amino acid analyses confirm the success of the procedures, showing quantitative yields of S-carboxymethylcysteine. The study also demonstrates the use of the reduced and carboxymethylated phenylalanyl chain of insulin as a substrate to test the purity of trypsin. Chromatographic separation of peptides from enzymatic hydrolysis is performed using a 15-cm column of the amino acid analyzer. The results show that trypsin hydrolyzes arginyl bonds more slowly when X and Y carry negatively charged groups. The presence of cysteic acid residues reduces the susceptibility to trypsin action compared to S-carboxymethylcysteine residues. The study concludes that reduced and carboxymethylated proteins can be used for structural studies, provided that thioether sulfur of methionine and indole group of tryptophan are protected from oxidation during peptide separation. The methods described provide a reliable way to prepare and analyze proteins for structural studies.