Albumin titration curves

The titration curve of serum albumin (Tanford et ah, 1955b) is independent of time and essentially completely reversible. However, the protein undergoes at least three changes in conformation during the course of titration. These changes are themselves rapid and reversible, so that sepa-... 

As the titration curves for the various albumins show, a considerable amount of NBS is consumed before a steady decrease of e at 280 m/x sets in. Apparently other reactive groups, especially thiol groups, react faster with NBS than tryptophan in this case. 

Recently Tanford and his collaborators have made use of the electrostatic term w to analyze the titration curves of a number of proteins. By combining graphical methods and trial-and-error selection they obtained values of p(Kint) and w from experimental titration curves. In each case the value for the apparent average net charge was corrected for the binding of anions and cations as determined by independent experimental methods. Differences between their experimental values of w and those calculated from theory were interpreted by these workers in terms of alterations in molecular parameters or structure. For example, observed values which were low, especially when they became low only at pH s far removed from neutrality, were attributed to alteration in the size or shape of the molecule brought about by electrostatic repulsion. Thus, with human serum albumin Tanford (1950) found that although the basic branch of the titration curve could be computed from theory, the acid branch was much too steep... 

The electrophoretic data of Alberty and Marvin (1951) on bovine serum albumin, unlike those discussed above, were obtained in unbuffered solutions. They show that chloride ion binding increases as the solution becomes more acid the indicated net charge on the protein is therefore less than that predicted from the titration curve. The extent of chloride binding found by these authors agrees well ivith that determined by Scatchard, Scheinberg, and Armstrong (1950a) for human serum albumin from dialysis equilibrium and electromotive force data. 

Phase boundaries were also developed for p-lactoglobulin, chicken egg albumin, lysozyme, ribonuclease, and trypsin, all at r=100, a weight ratio at which polymer saturation appears to take place (see Discussion section). For each protein, pHcritical was converted to net negative surface charge (Zpr) per unit protein surface area (A), using potentiometric titration curves (26-31) and hydrodynamic radii (32) found in literature. Plots of surface charge density (Zpr/A) vs. I are shown in Figure 3. 

The titration curves of a phosphorylated serum albumin preparation showed an additional 60 X 10 equivalent acid groups per gram protein in the region of pH 2.0-2.5 and a similar increase between pH 5.0 and pH 8.0. Since there had been an increase of 56 X 10 equivalents of phosphorus per gram protein in this preparation, the authors point out that this increase of two titratable equivalents per phosphorus means that most of the phosphorus is linked to the protein as a mono ester. 

The titration curve of formolized egg albumin deviates from that of normal protein in the region of pH 6-10, and Kekwick and Cannan (237) have indicated that of all the titratable groups only the amino groups combine. 

Fig. 17. Titration data for the carboxyl groups of bovine serum albumin, at 25°C and ionic strengths 0.01 (lowest curve), 0.03, 0.08, and 0.15 (upper curve), plotted according to Eq. (14) (Tanford et al., 1955b).

Figure 17 shows an even more striking anomaly. It represents the titration of the carboxyl groups of bovine serum albumin (Tanford et al., 1955b), and it is seen that a constant value of w will not fit the data at all, the plots of pH — log[a /(l — a )] versus Z being curved. It should be noted however that the steepest parts of the plots (near Z = 0) are linear, and that the slopes give w values of 0.054, 0.036, 0.028, and 0.023, respec-... 

The conversion of -ammonium groups to guanidinium by such reagents as 0-methylisourea results in a substantial elevation of their pK, and thus has been used for purposes similar to those just described in connection with formaldehyde. Tanford (1950) found that the titration of guani-dinated human serum albumin could be fitted by a curve computed on the assumption that all the e-ammonium groups had been changed to guanidinium, i.e., that below pH 12 only the readily separable dissociations of histidine and tyrosine were involved. 

---