On the reaction of papain and succinylpapin with diazo-1-H-tetrazole.
1. The reaction of papain and succinylpapain with diazo-1-H-tetrazole was investigated under different conditions. The extent of modification of the amino acids histidine, tyrosine, tryptophan and lysine was determined spectrophotometrically and/or by amino acid analysis. 2. Only one of the two histidine residues present in the enzyme reacts with diazo-1-H-tetrazole forming a monoazo derivative. The pH dependence of the coupling reaction reveals a normal pK of this reactive histidine. There are several arguments suggesting that this may be histidine 159 near the essential SH-group of papain. 3. All five tryptophan residues of the protein react with the diazonium ion below pH 7 forming a monoazo derivative with an absorption maximum at 370 nm, above pH 7 only four residues couple with diazo-1-H-tetrazole. The reaction of one tryptophan and one histidine are correlated as can be concluded from the pH dependence of the coupling rate of both amino acids and the parallel impairment of the catalytic acitivity. 4. 10-11 tyrosine residues out of 19 react with diazo-1-H-tetrazole to give bisazo compounds. 5 residues involved in hydrogen bridges form monoazo compounds. Only 12 tyrosines can be acylated by acetylimidazole. A relationship between the extent of modification of tyrosine and the activity of the enzyme could not be found.
D000596:Amino Acids / Q000032:analysis; D001393:Azoles; D001665:Binding Sites; D003979:Diazonium Compounds; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010206:Papain; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013386:Succinates; D013438:Sulfhydryl Compounds / Q000032:analysis; D013777:Tetrazoles; D014508:Urea
D000596:Amino Acids; D001393:Azoles; D003979:Diazonium Compounds; D013386:Succinates; D013438:Sulfhydryl Compounds; D013777:Tetrazoles; D014508:Urea; D010206:Papain