Spectroscopic Investigation of the Reaction of Sperm Whale Myoglobin with Zinc.

The relationship of macromolecular structure to biological activity will be understood in its totality only when complete structural determinations of representative macromolecules have been made. But even then, as now, a variety of physicoand biochemical methods will necessarily be brought to bear on such problems as the nature of the forces which determine and maintain structural integrity and the effect of distribution, spatial arrangement, and state of the various reactive groups in a protein molecule upon structure and biological activity. These experiments are naturally of interest for molecular genetics and the biosynthesis of proteins because they provide, among other things, information concerning the extent to which the amino acid sequence of a polypeptide chain determines per se its state of folding in a globular protein. Explanation, for example, of how such a slight change in primary structure as replacement of a single glutamic acid residue in hemoglobin A by valine in hemoglobin S can so profoundly modify the physiological activity of the protein, will be a major advance in our understanding of life processes. One approach to these problems is the study of complex formation between metallic cations and proteins. In fact, because of their widespread occurrence and important biological activities, metal-protein complexes constitute in themselves a problem of considerable interest. ' Provocative problems in this area are concerned with the nature of the heme-heme interaction in hemoglobin and the possible involvement of sulfhydryl groups in its mechanism; unmasking of metal binding sites on some protein molecules by denaturation or, as in the case of the interaction of Zn++ with serum albumin, simply by raising the temperature from 00 to 370C; stabilization of some proteins to enzymatic digestion or heat by metal ions; structure and mechanisms of action of metalloenzymes; role of Ngg++ in the structural stability of ribosomes; and binding of metal ions into chelate structures as in the case of complexes of hemoglobin with Hg++, (Fe+++)2-conalbumin, and Zn±++insulin. The specific and reversible combination of Zn++ with a variety of proteins often results in insoluble Zn++-protein complexes. This fact underlies a system of fractionation of the plasma proteins2 and a method of separating fetuin from the other proteins of fetal serum.3 It has now been demonstrated spectroscopically that combination of Zn++ with myoglobin causes reversible alterations in macromolecular conformation. Mlyoglobin is an ideal protein for such investigations since its exact three-dimensional structure has been largely established by the crystallographic X-ray analyses of Kendrew and co-workers,4 thereby providing the fundamental information required for a detailed description of the mechanisms of its reactions. The realization that binding of Zn++ by myoglobin mediates conformational changes has important implications, not only for methods of fractionation of biological materials, but also for the mechanisms of biochemical reactions such as the metal-activation and poisoning of enzymes.