Comparison of sperm whale metmyoglobin with its guanidinated derivative.

The description of the conformationof sperm whalemetmyoglobin in the crystalline state by Kendrew et al. (2,3) opens the way for correlating information about the crystalline structure with evidence obtained in solution. The reactivity of functional groups in the protein in solution towards various reagents may offer insight as to their availability and hence indicate the degree to which the groups are exposed to the solvent or taken up in internal interactions that stabilize the structure of the protein (4, 5). This indirect information may be correlated with the direct information from the crystallographic analysis. The reaction that we have chosen for a study of reactivity and for the correlation of the properties of the modified and unmodified protein is guanidination by reaction of lysyl residues with 0-methylisourea to yield homoarginyl residues without disruption of the peptide linkages (6-9). The homoarginyl as well as the fysyl residues are readily measured by standard analytical procedures (10). Because the conversion of amino into guanidino groups has little effect on the formal electrostatic charge of the protein in the pH region below 9, the disruptive effects of high net charge are avoided. Knowledge of the properties of model compounds is sufficient that some of the simpler consequences of guanidination can be searched for directly and sometimes interpreted with assurance (1, 11, 12). In the present study it is found that sperm whale metmyoglobin undergoes essentially complete conversion of lysyl into homoarginyl residues. Some variation in the reactivity of individual lysyl residues is indicated by the results to be reported, but a clear separation into classes according to reactivity is not established. The generally ready and complete reactivity of the protein has made it possible to give most attention to the second principal aim mentioned earlier, that of comparison of guanidinated with unmodified metmyoglobin. This comparison is made in terms of hydrodynamic properties, titration, reactivity towards p-nitrophenyl acetate and bromoacetate, and general stability. The way to a further correlation with structural information is opened by the preparation of the guanidinated protein in crystalline form.