Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis.
A general method by polyacrylamide gel electrophoresis for the determination of sulfhydryls and disulfides in a protein was developed. The method included a two-step alkylation procedure: the first step consisted of alkylation of the sulfhydryl groups with iodoacetic acid in the presence and absence of 8 M urea; the second step consisted of alkylation of the disulfide groups with iodoacetamide after reduction with a thiol. By high-pH urea gel electrophoresis, all the half-cystine residues in a protein could be categorized into three states: reactive sulfhydryls, nonreactive sulfhydryls, and disulfide bonded. The particular advantage of the method is that the states of half-cystines in different protein species can be analyzed independently both in isolated protein and in biological translation systems.
Takahashi
N
N
Research Institute for Food Science, Kyoto University, Japan.
Hirose
M
M
D000478:Alkylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide; D007461:Iodoacetates; D019807:Iodoacetic Acid; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010084:Oxidation-Reduction; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry*; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry; D014508:Urea
D004220:Disulfides; D007461:Iodoacetates; D011506:Proteins; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; D013879:Thioredoxins; D014508:Urea; D010047:Ovalbumin; D019807:Iodoacetic Acid; D007460:Iodoacetamide