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The reactions of horse globin reconstituted with proto-, deutero-, and mesoheme have been examined by equilibrium and kinetic methods. In virtually all reactions studied, mesohemoglobin displays the more extreme functional behavior, whereas deuterohemoglobin exhibits behavior which is either very similar to native hemoglobin or intermediate between the two. Our kinetic and equilibrium results indicate that the primary effect of heme modification on the functional properties of hemoglobin is to alter the intrinsic reactivities of the deoxy and liganded conformations. Heme modification does not, however, result in substantial alterations in the conformational equilibrium between the two states. Simple inductive electronic effects of the 2- and 4-substituents of the heme moiety in deutero- and mesohemoglobin are apparently not sufficient to explain the observed equilibrium and kinetic properties completely, which indicates that steric effects of these substituents may also play a role in determining the functional behavior of the hemoglobin molecule.

A new purification scheme for both prolactin and growth hormone from bovine pituitaries has been developed which avoids the use of potentially damaging solution conditions. Both hormones were greater than 95% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had specific activities similar to or greater than standard samples of the same hormone as judged by several bioassays. Small single crystals of bovine growth hormone were obtained by vapor diffusion techniques. Examination of these crystals by x-ray diffraction, using the Cornell High Energy Synchrotron Source, showed that they were well ordered, and exhibited diffraction to 2.8-A resolution on still photographs. Precession and oscillation photographs showed that they belonged to the orthorhombic space group P2(1)2(1)2(1) (or P2(1)2(1)2) with unit cell dimensions a = 219 A, b = 51.9 A, c = 68.9 A. The density of the crystals was 1.19 +/- 0.02 g/ml from which the presence of eight 45,000-dalton dimers/unit cell was deduced. The protein content of the crystals was shown by isoelectric focusing to be identical to that of purified growth hormone in solution. These crystals appear suitable for use in the x-ray structure determination of bovine growth hormone to at least 3.2-A resolution.

DesHis (146β) hemoglobin is a β chain modification of human hemoglobin in which the COOH-terminal histidine 146β has been removed by digestion with carboxypeptidase B. Previous crystallographic investigations of the structures of desHis deoxyhemoglobin and deoxyhemoglobin Hiroshima (His 146β → Asp) suggest that the atomic structures of these derivatives differ from each other and from hemoglobin A only in the immediate environment of the COOH-terminal residue. Kinetic studies of the binding of oxygen, carbon monoxide, and n-butyl isocyanide to desHis hemoglobin reveal that the properties of desHis hemoglobin and hemoglobin Hiroshima are indeed similar, but not identical; the properties of both differ appreciably from those of hemoglobin A. These qualitative differences in kinetic properties are not readily reconciled with the apparently minor structural differences revealed in the structural studies. They suggest rather that the unliganded forms of both derivatives adopt a new conformation, or series of conformations in slow equilibrium, which are distinct from the conformations of either deoxy- or oxyhemoglobin A.

Dissociation of both human deoxyhemoglobin (deoxy-Hb) and carboxyhemoglobin (HbCO) at alkaline pH exposes previously buried tyrosine residues to the environment and alters their pK. Subunit association and dissociation of deoxy-Hb and HbCO can be followed through spectral changes which arise from these changes in tyrosine ionization above pH 10.0. The difference spectrum of the dissociation reaction contains a contribution from both tyrosine and tryptophan. Ultracentrifuge results confirm that a tetramerdimer equilibrium exists at these high pH values. The tetramer-dimer dissociation constant, K4,2, of both HbCO and deoxy-Hb increases dramatically above pH 10.0 and the value of K4,2 for HbCO is significantly greater than the value of K4,2 for deoxy-Hb in the pH range of 7 to 11. The groups responsible for the spectral change on dissociation have been tentatively identified with Tyr C7(42)α and Trp C3(37)β, which lie in the α1-β2 interface. Substantial quantities of deoxy-Hb dimers are produced at high pH. The properties of these dimers have been examined both at high pH and at pH 7.0 after rapid pH drop in the stopped flow apparatus. Association of deoxy-Hb dimers to the tetramer is accompanied by a spectral change in the Soret region, which has allowed us to follow the association reaction at pH 7.0. Deoxy-Hb dimers react rapidly with CO at a rate characteristic of noncooperative species (l' = 6.5 x 106 m-1 sec-1). Further, human haptoglobin 1-1 binds these deoxy-Hb dimers at pH 7.0 at a rate similar to that observed with HbCO dimers (6.3 x 105 m-1 sec-1 and 5.5 x 105 m-1 sec-1, respectively). We conclude that the deoxy-Hb dimer, the unliganded dimer derived from liganded hemoglobin, and the HbCO dimer are identical in conformation, α1β1; the first two are noncooperative in their ligand-binding properties. The hemoglobin tetramer is therefore the minimum unit capable of full cooperativity in ligand binding. Models in which the free hemoglobin dimer is assumed to possess basically the same functional properties as the tetramer are untenable.