pH‐jump titration of the tyrosyl groups of bovine plasma albumin

Abstract

SynopsisIonization properties of the tyrosyl groups of bovine plasma albumin in various conformational states-the native state (N), the two acid states (F and E), and the state (B) stable at slightly alkaline pH-were studied by means of a stopped-flow pH-jump technique. The technique allows us to obtain the tyrosyl titration curve in a conformational state that is unstable in the pH region of the titration. The pH jumps from the N and B states to various alkaline pH's, where the tyrosines ionize to bring about a timedependent increase in absorption at 296 nm, indicating that a number of the tyrosines buried initially become susceptible to ionization as a result of the alkaline transition occurring above pH 10.8. Extrapolation of the observed absorption change to zero time gives a spectrophotometric titration curve in the initial conformational state. Only 3040% of the 19 tyrosines of the protein can ionize both in the N and the B state a t pH 12. The pH jumps from the F and E states, on the other hand, give a decrease in absorption between pH 9 and 11.7, indicating that the tyrosyl groups initially exposed are remasked by refolding after the pH jumps, and the zero-time titration curves show that essentially all the tyrosyl groups ionize normally in these acid states. The results are discussed in relation to the known results of the tyrosyl exposure of the protein measured by other techniques, and the consistency among them demonstrates the effectiveness of the pH-jump titration method. Hydrogen bonding between the abnormal tyrosyl and carboxylate groups as'a mechanism to stabilize native albumin is suggested from characteristics of the alkaline transition, which also involves the exposure of the tyrosyl groups, and from the tyrosyl titration curves in the native and acid states.