Proton-dependent dissociation equilibrium of hemoglobin. 2. Surface pressure measurements in monolayers of horse hemoglobin(III)

Abstract

The molecular weight of hemoglobin (III) in monolayers on aqueous subsolutions has been determined by measuring the surface pressure as a function of the protein surface concentration. The dissociation equilibrium between tetrameric and dimeric hemoglobin (III) was determined for spread as well as adsorbed monolayers. The results were compared with analogous measurements in solution. It was found that the numerical value and the pH dependence of the dissociation constant were similar both in the bulk and in the surface phase of the solution. From these findings it was concluded that the native conformation of hemoglobin (III) is retained after adsorption at aqueous surface.

Effect of ionization on the monolayers of bovine serum albumin, hemoglobin, and insulin at the air-water interface

“…The protein is reported to be in tetrameric form in this pH range in bulk. 17 Using the tetramer molecular weight as 68000, we find that = kT (as -*• 0) on subphases with pH 7.4 (isoelectric pH18). This result thus indicates that the effect of subphase pH on the number of charges in the protein molecule can be analyzed from these vs. plots, as described in further detail below.…”

“…In contrast, the current literature studies reported on such charged protein films, at air-water interfaces, do not take into account the repulsion term IIe. 16,17 In these reports the relation given by eq 3 is used. Accordingly, these investigators thus report molecular weight of BSA to be 17 300, on subphase of pH 3.9.16 We will argue that this conclusion is in error, since the BSA molecule is stable at this pH in bulk.…”