Kinetic Characterisation of the R and R2 Quaternary Structures of Oxyhemoglobins Arising from Different Effects of Inositol Hexakisphosphate on Their Reactions with Ellman's Reagent.

In a previously reported equilibrium study of the reaction of 5,5'-dithiobis(2-nitrobenzoate), DTNB, with various carbonmonoxyhemoglobins over the pH range 5.6 to 9, we obtained contradictory results on the influence of the allosteric effector inositol hexakisphosphate (inositol-P) on the DTNB reaction. For this reason, we replaced the carbonmonoxyhemoglobins with oxyhemoglobins and investigated the effect of inositol-P on the equilibrium and of their reactions with DTNB over the same pH range.

Quantitative characterization of non-specific interaction of two globular proteins with Dextran T70 in a binary mixture.

In a bid to quantify the contribution of molecular structure to non-specific interactions leading to functionally important structural changes in cellular processes, the self-interaction of dextran-T70 (DT70) and its interaction with each of bovine serum albumin (BSA) and ovomucoid trypsin inhibitor (OVO) were studied at pH 7.4 between 5 and 37 °C. The dependences of the apparent molecular weight of each of BSA, OVO and DT70 on the concentration of DT70 were independent of temperature.

Catalytic studies of glutathione transferase from Clarias gariepinus (Burchell) in dilute and crowded solutions.

Kinetic properties of purified Clarias gariepinus glutathione transferase (CgGST) was studied in the presence of Ficoll 70, Polyethylene glycol (PEG) 6000, bovine serum albumin (BSA) and in dilute solution. This was done to mimic the cytosol thereby unraveling the actual mechanism of detoxication involving glutathione transferase (GST) in the crowded intracellular milieu. CgGST from the liver of Clarias gariepinus was purified to homogeneity by affinity chromatography on glutathione (GSH) - agarose.

Denaturation studies of Clarias gariepinus glutathione transferase in dilute and crowded solutions.

It is important to understand the effect of crowding conditions on the native structure and functional state of enzymes. Equilibrium denaturation studies of Clarius gariepinus GST (CgGST) by guanidine hydrochloride (GdHCl) under dilute conditions and in separate solutions of 0-100 g dm Ficoll 70, polyethylene glycol 6000 (PEG 6000) and equal w/v mixtures of the two polymers at 25 °C and pH 7.4 were studied fluorometrically.

Quantitative characterization of the concentration-dependent interaction between molecules of Dextran 70 in aqueous solution: Measurement and analysis in the context of thermodynamic and compressible sphere models.

The static light scattering and sedimentation equilibrium of solutions of Dextran 70 were measured as functions of concentration up to 100 g/L in pH 7.4 phosphate-buffered saline at temperatures between 5 and 37 °C. The concentration dependence of scattering intensity and the apparent molar mass obtained from sedimentation equilibrium were found to be nearly independent of temperature over this range to within the uncertainty of measurement.

R and R2 quaternary structures of carbonmonoxyhemoglobins: Differential effect of inositol hexakisphosphate on their affinity for Ellman's reagent.

The reaction of 5,5'-dithiobis(2-nitrobenzoate), DTNB, with hemoglobin sulfhydryl groups is linked to three negatively contributing Bohr effect groups: His2β is present in all avian hemoglobins but absent in some mammalian hemoglobins; His77β and His143β are absent in avian but present in nearly all mammalian hemoglobins. To probe the consequences of these differences, we determined the influence of inositol hexakisphosphate (inositol-P) on the DTNB affinities of avian and mammalian carbonmonoxyhemoglobins. Inositol-Pdecreases by two orders of magnitude the DTNB affinity of guinea pig hemoglobin, which has His2β and the R2 quaternary structure.

Quantitative characterization of temperature-independent polymer-polymer interaction and temperature-dependent protein-protein and protein-polymer interactions in concentrated polymer solutions.

To study the effect of non-specific interactions arising from proteins being in a crowded environment on physiological processes, the self-interaction of concentrated Dextran T70 and Ficoll 70 and the interactions between a dilute protein and these polymeric macromolecules were quantified using non-ideal tracer sedimentation equilibrium. Sedimentation equilibria of each polymer were measured between 5 and 37 °C, and sedimentation equilibria of 2 mg cm superoxide dismutase (SOD) in 0-0.1 g cm of each polymer was also measured.

Bohr effect of human hemoglobin A: magnitude of negative contributions determined by the equilibrium between two tertiary structures.

We have measured the affinity of the CysF9[93]β sulfhydryl group of human deoxyhemoglobin and oxyhemoglobin for 5,5'-dithiobis(2-nitrobenzoate), DTNB, between pH ≈5.6 and 9 in order to understand the basis of the reported reduction of the Bohr effect induced by chemical modification of the sulfhydryl. We analyzed the results quantitatively on the basis of published data indicating that the sulfhydryl exists in two conformations that are coupled to the transition between two tertiary structures of hemoglobin in dynamic equilibrium.

Quantitative characterization of temperature-independent and temperature-dependent protein-protein interactions in highly nonideal solutions.

The interaction among each of three dilute "tracer" proteins (bovine serum albumin, superoxide dismutase, and ovomucoid) at a concentration of 2 mg/mL and each of two "crowder" proteins (ovomucoid and BSA) at concentrations up to 100 mg/mL was characterized by analysis of dependence of the equilibrium gradients of both tracer and crowder upon the concentration of crowder. The equilibrium gradients of both crowder proteins were found to be independent of temperature over the range 5-37 °C. The equilibrium gradients of tracer BSA and ovomucoid in the complementary crowder species were likewise found to be independent of temperature over this range, indicating that interaction among these tracers and crowders is predominantly repulsive and essentially entirely entropic in nature.

Quantitative characterization of polymer-polymer, protein-protein, and polymer-protein interaction via tracer sedimentation equilibrium.

Quantitative analysis of the composition dependence of the concentration gradient of each species of macromolecule within a solution mixture at sedimentation equilibrium permits the quantitative characterization of self- and heterointeractions between sedimenting solutes. Sedimentation equilibrium experiments were conducted on solutions containing a trace concentration of FITC-labeled BSA in varying concentrations of Ficoll 70 and on solutions containing a trace concentration of FITC-labeled Ficoll 70 in varying concentrations of BSA. The equilibrium gradient of each solute component in each mixture was measured independently.

Transition of hemoglobin between two tertiary conformations: determination of equilibrium and thermodynamic parameters from the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group.

The equilibrium constant of the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of hemoglobin decreases by 2 to 3 orders of magnitude between pH 5.6 and 9. The reaction is coupled to the ionizations of two groups on the protein.

Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat: variation of the equilibrium and reverse rate constants with pH.

We have determined for the first time the equilibrium constant, Keq, for the reaction of Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate), with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat. In the pH range 5.6 to 9.

Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: acetylation of NH3+ terminal group of the beta chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form.

We demonstrate kinetically that the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3+ terminal group of GlyNA1[1]beta is free, kf, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH3+ terminal group of SerNA1[1]beta is acetylated, and the pH dependence profile of kf is simple.