The article discusses the preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins, focusing on the methods for cleaving disulfide bonds and subsequent alkylation of the resulting sulfhydryl groups. The authors compare the effectiveness of mercaptoethanol and sodium borohydride for reducing disulfide bonds, finding that mercaptoethanol is more satisfactory due to its lower tendency to cause side reactions. They detail the procedures for preparing reduced and carboxymethylated proteins, including the use of mercaptoethanol in 8 m urea and iodoacetate for alkylation. The article also describes improvements in the hydrolysis conditions to minimize side reactions and enhance the accuracy of amino acid analysis. Additionally, it presents methods for testing the purity of trypsin using the RCM-phenylalanyl chain of insulin and compares the rates of tryptic hydrolysis of performic acid-oxidized and reduced carboxymethylated ribonuclease. The study highlights the importance of protecting methionine and tryptophan residues from oxidation during chromatographic separation and provides insights into the structural studies of proteins.The article discusses the preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins, focusing on the methods for cleaving disulfide bonds and subsequent alkylation of the resulting sulfhydryl groups. The authors compare the effectiveness of mercaptoethanol and sodium borohydride for reducing disulfide bonds, finding that mercaptoethanol is more satisfactory due to its lower tendency to cause side reactions. They detail the procedures for preparing reduced and carboxymethylated proteins, including the use of mercaptoethanol in 8 m urea and iodoacetate for alkylation. The article also describes improvements in the hydrolysis conditions to minimize side reactions and enhance the accuracy of amino acid analysis. Additionally, it presents methods for testing the purity of trypsin using the RCM-phenylalanyl chain of insulin and compares the rates of tryptic hydrolysis of performic acid-oxidized and reduced carboxymethylated ribonuclease. The study highlights the importance of protecting methionine and tryptophan residues from oxidation during chromatographic separation and provides insights into the structural studies of proteins.