States of amino acid residues in proteins. XVIII. A revised way of using diazonium- i-H-tetrazole for reactivity examination of histidine and tyrosine residues

Abstract

Diazonium- i-H-tetrazole explored previously for discrimination of various states of histidine residues in proteins affords spectrally different bisazo derivatives of histidine and tyrosine, having a visible absorption band at 480 and 548 mμ, respectively. The overlapping effects of monoazotyrosine and bisazotyrosine bands on the bisazohistidine band was neglected in the previous method, and this approximation was pointed out later to include considerable errors. Corrections were made in the present study, taking account of all the mutual overlapping effects of the visible bands of bisazohistidine, monoazotyrosine and bisazotyrosine, and revision was made in the corrected method to determine the concentrations of these three derivatives of histidine and tyrosine residues. Application of the revised method to insulin revealed the following facts which indicate that the overlapping effects are properly corrected in the revised photometric method. One of the total two histidine residues in the insulin molecule is biscoupled, and the other remains as the monoazo derivative. Three of the four tyrosine residues are biscoupled, and the other one residue remains as the monoazo derivative. The two α-amino groups at A and B peptide termini and the ε-amino group of B29 Lys are modified with the rates in the order, A1 Gly > B1 Phe > B29 Lys.