Proteins as buffers.

It was a strange choice to ask me, who has not contributed to the description of the titration behavior of proteins, to discuss tha t subject. Nevertheless I am pleased to attempt to summarize the state of knowledge for our present purposes. Our best objective in this monograph is surely to make ourselves and each other better informed; we will be examining the meaning of terms for that purpose and not, I hope, t o attempt to legislate their use. I n that spirit the present assignment certainly promises to be informative to me. Buffering refers to the minimization of p H change on the addition of acid or alkali. We evaluate the extent of buffering by seeing how much acid or alkali is required to change the pH; tha t is, by titrating. The steepness of the titration curve a t any point yields the buffer value a t tha t pH. About onethird of the amino acid residues in proteins contribute titratable groups; the number of such groups in many common proteins ranges from about 20 to over 200. The common groups include the carboxyl groups, contributed by the sidechains of glutamic and aspartic acid; the amino groups on lysine sidechains; the guanidinium groups contributed by arginine sidechains; the imidazolium groups of histidyl residues; the phenolic groups contributed by tyrosyl residues; and the sulfhydryl groups of cysteine residues. Hemoglobin contains also the propionate sidechains of heme, and an acidic water molecule on each iron atom. In addition, nearly all proteins contain terminal a-amino and a-carboxyl groups on their polypeptide chains. FIGURE 1 shows a titration curve obtained for p-lactoglobulin by Nozaki.’ The ordinate label illustrates that the titratable groups may be counted from any desired reference point. The figure shows how the titration curve may be divided into regions; in this case the titration has been terminated before the guanidinium groups were deprotonated, or we should see a fourth region. The groups titrating in the acid range may tentatively be identified as carboxyl groups, those in the middle range as imidazole and terminal amino groups, and those titrating between p H 8.5 and 11.5 as sidechain amino, phenolic and sulfhydryl groups. Independent determinations of the number of terminal carboxyl and amino groups permit subtraction of these quantities from the first and second titration regions, respectively, where each is likely to be titrated, to give us a presumptive count of sidechain carboxyl and imidazole groups. We may then determine how many phenolic groups have been titrated in the upper range, by performing a separate spectro-photometric titration; that is by observing the location and extent of a spectral change at 295 mb