The Reaction of Amino Acids and Proteins with Diazonium Compounds. I. A Spectrophotometric Study of Azo-Derivatives of Histidine and Tyrosine.

Abstract

The coupling in alkaline solution of free histidine and tyrosine with p-diazobenzene-sulphonic acid has been studied as a basis for quantitative investigation of the behaviour of proteins in the presence of diazonium compounds. Both mono- and bisazo- derivatives may be formed with each amino acid, the wavelengths of maximum absorption being 380 and 490 mμ respectively for histidine and 325 and 480 mμ respectively for tyrosine ; the wavelength displacements on accepting a second diazonium group are comparable to effects reported for similarly conjugated systems. When appropriate corrections are made for other absorbing components, the extinctions at 490 mμ and the initial histidine concentrations up to the (bis) equivalent of diazonium compound conform to Beer's law, showing that bis(azobenzenesu1phonic acid)histidine is formed exclusively under these conditions. At higher histidine concentrations mono(azobenzenesulphonic acid)histidine is formed in increasing proportions. Even at low initial tyrosine concentrations both mono- and bis(azobenzenesulphonic acid)-tyrosine are formed, although mono-coupling is again favoured more as the concentration increases. No direct reaction between histidine and the nitrous acid used in the formation of the diazonium compound could be detected spectrophotometrically, but diazotization of tyrosine appears to reduce the propensity for bis-coupling to some extent. If urea is incorporated in the system to destroy excess nitrous acid, a yellow compound having maximum absorption at 430 mμ is formed between p-diazo-benzenesulphonic acid and the urea.