Preparation and characterization of sulfanilazo and arsanilazo proteins

• Published in: Biochemistry (Easton) Vol. 23; no. 4; pp. 596 - 603
• Main Authors: Pielak, Gary J, Urdea, Mickey S, Legg, J. Ivan
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.01.1984
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; derivatives; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; histidine; Proteins; tyrosine; Diazotization; Proteins; PH; Chemical modification; Preparation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00299a003

A thorough study of the susceptibility of a series of proteins to azo coupling as a function of pH, ratio of diazonium salt to protein, and type of diazonium salt was undertaken. Spectral analysis of the azo proteins indicates that tyrosine is modified in preference to histidine, that while both possible structural isomers of azohistidine can be formed, azo coupling at the C-2 position is more common, and that little if any bis coupling occurs. Use of diazotized ( sub(u35)S)sulfanilic acid indicates that the majority of residues modified in azo proteins is not detected byspectral analysis. These residues were identified as the products of the reaction of diazonium salt with free amines (i.e., N-terminal and/or the epsilon -amine of lysines) and with sulfhydryl groups. The former reactions are decreased or eliminated by acylation prior to azo coupling of the protein. The extent of azo coupling was found to be characteristic of the particular protein and not simply a function of the total number of potentially reactive residues in a protein.