Laura D. Kwiatkowski

Functional properties of hemoglobins from deep-sea fish: correlations with depth distribution and presence of a swimbladder

The ligand binding properties of the hemoglobins of several deep-sea, bottom-living fish have been examined. These include five species of rattails (Macrouridae) and Antimora rostrata, all of which possess swimbladders, and two unrelated species without swimbladders, Bathysaurus mollis and Alepocephalus sp. All of the hemolysates of these fish exhibited the Root effect with a minimum ligand affinity at about pH 6 in the presence of organic phosphate. Under these conditions the hemolysates from fish which possess swimbladders exhibit two roughly equal populations of heme groups with markedly different ligand affinities. For the deeper-dwelling species the affinities for carbon monoxide differ by some 500-fold, the low-affinity population having a p50(CO) of 100 mmHg at 15 degrees C. This very low affinity is associated with a second-order rate constant for CO combination of the order of 10(3) M-1 X s-1. Those species without swimbladders have hemoglobins which do not have such heterogeneous binding sites, suggesting a relationship between these very-low-affinity heme groups and the pumping of oxygen into a swimbladder at high hydrostatic pressures.

On the role of superoxide radical in the mechanism of action of galactose oxidase.

Abstract The inhibition of galactose oxidase by superoxide dismutase is a function of the method of assay, nature of substrate, and composition of incubation and assay mixtures, as well as the concentration of dismutase. A reasonable level of inhibition is attained only when superoxide dismutase is present prior to the onset of catalysis although this effect is not observed under all conditions tried. Peroxidase activates galactose oxidase and blocks its interaction with either superoxide dismutase or catalase. These results further obscure the possible role of superoxide radical in the galactose oxidase reaction.

STRUCTURE AND OXYGEN AFFINITY OF CRYSTALLINE DES-HIS-146BETA HUMAN HEMOGLOBIN IN THE T STATE

To correlate directly structure with function, the oxygen affinity and the three-dimensional structure of crystals of the T quaternary state of des-His-146β human hemoglobin have been determined by polarized absorption microspectrophotometry and x-ray diffraction crystallography. In des-His-146β, the COOH-terminal histidine residues of the β chains of hemoglobin A have been removed. Oxygen binding to crystalline des-His hemoglobin is non-cooperative and independent of pH. The oxygen affinity is 1.7-fold greater than that of the crystalline state of hemoglobin A. Removal of His-146β results in a small movement of the truncated COOH-terminal peptide and in a very small change in quaternary structure. Previously, similar studies on T state crystals of des-Arg-141α hemoglobin showed that removal of the COOH termini of the α chains results in much larger effects on oxygen affinity and on quaternary structure. Kinetic studies in solution reveal that at pH 7.0, the rates of CO combination with deoxygenated des-His-146β in the absence and presence of inositol hexaphosphate are 2.5- and 1.3-fold, respectively, more rapid than for hemoglobin A. The values for des-Arg are 7.6- and 3.9-fold. The properties of the T state of hemoglobin both in the crystal and in solution are influenced to a greater degree by the interactions associated with Arg-141α than those associated with His-146β.

[9] Fish hemoglobins

Publisher Summary Fish hemoglobins exhibit the essential features of mammalian hemoglobins, cooperative ligand binding and heterotropic responses to a variety of ionic species, but they display an astounding variety of functional behaviors. These different properties are of interest as examples of evolutionary adaptation to differing physiological and environmental needs. They also offer valuable systems in which to study specific phenomena in either an exaggerated form or a simplified context. There are components of the hemoglobins of Salmonidae that exhibit neither Bohr effects nor responses to organic phosphates. Many fish hemoglobins exhibit exaggerated Bohr effects, commonly known as the “Root effect,” permitting the study of pH dependencies, which are considerably amplified relative to that observed in mammalian hemoglobins. Fish hemoglobins can exhibit widely differing ligand affinities, with the total range reported for different hemoglobins under varying conditions being greater than four orders of magnitude. Additionally, many fish hemoglobins that exhibit the Root effect lose cooperative ligand binding when they attain their minimum ligand affinity at low pH in the presence of organic phosphates. These noncooperative, low-affinity states appear to be excellent models of liganded T states and are ideal for the analysis of the origins of the differences in the ligand affinities of the two extreme quaternary states of hemoglobin—namely, the R and T states.

Kinetic mechanism of the Cu(II) enzyme galactose oxidase

The steady-state kinetics of four redox reactions catalyzed by galactose oxidase have been determined. The alcohol substrate used in each case was galactose; the four oxidant substrates used were O2, IrCl6(2)-, porphyrexide, and Fe(CN)6(3)-. With the exception of the last reagent, saturation behavior is exhibited by all substrates. Double reciprocal plots of rate data obtained varying one substrate at various concentrations of the other are intersecting for all parsi that exhibited saturation behavior. Thus, these reactions are kinetically sequential processes involving single central complexes. These complexes involve enzyme, galactose, and one molecule of oxidant, whether or not the oxidant is a one- or two-electron acceptor. This result indicates that for one-electron oxidants, an enzyme.alcohol-derived radical species may exist as a transient prior to the reaction of the second electron equivalent of oxidant. A similar substrate radical.O2- transient is postulated in the reaction involving O2. The inhibition by H2O2 has also been studied in detail. H2O2 apparently binds to the enzyme at two sites. The nature of alcohol and O2 binding to the enzyme Cu(II) is discussed in light of these kinetic results.

Correlation of protein functional properties in the crystal and in solution: the case study of T-state hemoglobin.

The relevance of three‐dimensional structures of proteins, determined by X‐ray crystallography, is an important issue that is becoming even more critical in light of the Structural Genomics Initiative. As a case study, a detailed comparison of functional properties of the T quaternary states of genetically or chemically modified human hemoglobins (Hbs) in solution and in the crystal was performed. Oxygen affinities of Hbs in crystals correlate with the rate constants of their initial combination with carbon monoxide (CO) in solution, indicating that changes in ligand affinity caused by the modifications are similarly observed in both physical states.