Stopped-flow studies of spectral changes in human serum albumin following an alkaline pH jump.

Abstract

A stopped-flow technique was used to study the spectral changes occurring in albumin following a pH jump from 11.3 to 11.8 at 25 degrees C. Ultraviolet difference spectra between various albumin species participating in the process are reported. These spectra are similar in shape to the difference spectrum between the phenolate and phenolic form of tyrosine. At pH 11.3 one-third of the 18 tyrosine residues in albumin are deprotonated. At pH 11.8 two-thirds are deprotonated. The total reaction was analyzed as a multistep unimolecular consecutive process completed in four or more steps. Estimates were made of the number of tyrosine residues involved in the individual transitions. The first transition occurs with a rate constant greater than 300 s-1, in which 4.3 tyrosine residues deprotonate. The second transition occurs with a rate constant of 56.6 +/- 5.9 s-1, deprotonating 1.5 tyrosine residues. During the third (3.4 +/- 2.8 s-1) and following transitions (less than 0.3 s-1), which could not be reproducibly separated, 0.7 tyrosine residues deprotonate. The rates of deprotonation are inconsistent with simple diffusional dissociation of protons from the tyrosine residues and reflect exposure of tyrosines through conformational changes of albumin or dissociations of stably hydrogen-bonded tyrosines.