Circular dichroism, the unequal absorption of right and left circularly polarized light, is a manifestation of optical activity in the vicinity of absorption bands. To the experimental scientist interested in the conformation of macromolecules and in the sensitive response of optical activity to conformational alteration, it offers a relatively new and powerful means of understanding the environment of chromophoric residues. As a tool in the elucidation of electronic spectra, it should be useful to the theoretical scientist in identifying weakly allowed absorption bands as well as in providing rotational parameters which can be compared with the developing theory of optical activity. I have stressed application of circular dichroism, to experimental aspects of protein and nucleic acid conformation in solution. Much is still uncertain in particular quantitative details. However, even these early results shed new light and yield new information on the conformation of these molecules.
A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free a-and -globin chains (a', ft') and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the a losteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at hig dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For a-globin, the yield is about 50% because of incomplete combination with the heme-containing P chain. For -globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.In two previous papers (1, 2), we described the preparation and properties of isolated heme-free human globin chains (a", a°) and of two kinds of reassembly intermediates in which both kinds of chains are present but only half of the heme sites are filled (HFa and HFft molecules). The first paper demonstrated that the separated globin chains near neutral pH have different conformations, a" being substantially more disordered than a", in contrast to the separated heme-containing subunits, which are folded similarly, and in contrast also to the heme-free chains when bound together in dimeric apohemoglobin (a°,B°). Moreover, it was shown that, once separated, the globin chains do not readily recombine at neutral pH and accessible temperatures to form apohemoglobin. The second paper demonstrated that if either chain contains a heme, then the complementary chains do specifically recombine. During such a recombination, the disordered a-globin is refolded to the conformation it possesses in apohemoglobin. The coupled binding and refolding was termed an alloplex interactiont, to emphasizethe extensive refolding induced by a neighboring subunit.In future papers on this subject, the kinetics and mechanism of the alloplex interaction and the equilibrium of the HFa and HFB intermediates will be reported. The present paper shows that human hemoglobin can be reconstituted from separated globin chains and hemin by two different routes and that the reconstituted hemoglobin is similar or identical to native hemoglobin with respect to cooperativity in oxygen binding, the Bohr effect, and response to the allosteric effector 2,3-diphosphoglycerate (P-glycerate).In the course of our work on this problem, we found it necessary to modify several of our previous...
Circular-dichroism measurements of solutions of poly-L-tyrosine in the helical conformation at pH 11.2 reveal three ellipticity bands at 270, 248, and 224 µ. The band at 224 µ, which is the ro-TT* peptide transition characteristic of helical polypeptides, is negative in sign and indicates that the poly-L-tyrosine helix is right-handed. From the measured dichroism bands and an estimate of the contribution of an additional peptide transition at 190 µ, an optical rotatory dispersion curve was computed and compared with the experimentally measured values in the wavelength interval 500-225 µ. The agreement is sufficiently satisfactory to warrant the belief that the major transitions governing the optical rotation in the visible and near-ultraviolet spectrum have been considered. It is shown that the displacement of the negative trough of the rotation by several millimicrons from its usual position at 233 mg in right-handed a-helical polypeptides is the result of an overlap of Cotton effects. The rotational strength, Rh, of the 224-mg transition is only about one-third of the value for fully helical poly-a,L-glutamic acid, but it is likely that a side-chain-chromophore transition of opposite-sign rotational strength is partly responsible for the apparently low value of helix content. Measurements of the dichroism of a nonhelical form of poly-L-tyrosine at the same pH show a positive dichroism band at 225 mg and another at about 245 mg.
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