Inhibition of human hemoglobin autoxidation by sodium n-dodecyl sulphate

The effect of sodium n-dodecyl sulphate (SDS) on hemoglobin autoxidation was studied in the presence of a 100 mM phosphate buffer (pH 7.0) by different methods. These included spectrophotometry, fluorescence technique, cyclic voltametry, differential scanning calorimetry, and densitometry. Spectroscopic studies showed that SDS concentrations up to 1 mM increased deoxy-, decreases oxy-, and had no significant effect on the met- conformation of hemoglobin. Therefore, a SDS concentration up to 1 mM increased the deoxy form of hemoglobin as the folded, compact state and decreases the oxy conformation. The turbidity measurements and differential scanning calorimetry techniques indicated a more stable conformation for hemoglobin in the presence of SDS up to 1 mM. Electrochemical studies also confirmed a more difficult oxidation under these conditions. The induction of the deoxy form in the presence of SDS was confirmed by densitometry techniques. The compact structure of deoxyhemoglobin blocks the formation of met-conformation in low SDS concentrations.     

Binding of sodium n-dodecyl sulphate and protons to catalase

The binding of sodium n-dodecyl sulphate to catalase has been measured by equilibrium dialysis in the pH range 3.2 to 10.0. On the acid side of the isoelectric point (pH 5.4) the surfactant anions initially bind to cationic sites on the protein and subsequent binding is cooperative. At high pH on the alkaline side of the isoelectric point only cooperative binding is observed. The binding data have been combined with protein titration curves to calculate the Gibbs energies of formation of protein titration curves to calculate the Gibbs energies of formation of protein surfactant proton complexes. Contributions to the Gibbs energies of complex formation by surfactant and protein binding have been estimated. The average Gibbs energies of surfactant binding to specific cationic sites are ca. 28 kJ mol^?1 and for cooperative binding ca. 15 kJ mol^?1.     

A microacalorimetric study of the interaction between trypsin and sodium n-dodecyl sulphate.

1. The binding of sodium n-dodecyl sulphate to trypsin and reduced trypsin has been measured by equilibrium dialysis at pH 3.5 and 5.5. 2. At pH 3.5 trypsin specifically binds surfactant at low concentration, at higher concentrations co-operative binding occurs. 3. Reduction of trypsin destroys the specific binding sites at pH 3.5. 4. At pH 5.5 both trypsin and reduced trypsin show only co-operative binding. 5. The interaction of sodium n-dodecyl sulphate with trypsin, reduced, inhibited, and thermally denatured trypsins has been studied by microcalorimetry at 25 degrees C. 6. The microcalorimetric measurements have been used to estimate enthalpy changes (deltaHd) on unfolding of trypsin; deltaHd = 82 +/- 5 kJ-mol-1 at pH 3.5 and 128 +/- 5 kJ-mol-1 at pH 5.5. 7. The unfolding of trypsin follows a different thermochemical pathway to that of reduced trypsin.     

Interaction between sodium n-dodecyl sulphate and chemically modified bovine catalases

A series of catalases have been prepared in which a proportion of the carboxyl groups of glutamate and aspartate residues have been amidated with glycinamide. The physical properties of the amidated catalases have been investigated with specific reference to their interaction with sodium n-dodecyl sulphate (SDS). Amidation leads to an increase in SDS binding at pH 6.4. Microcalorimetric measurements show that the exothermic enthalpy of interaction with SDS increases with the extent of amidation in acid solution (pH 3.2–6.4). The increase in exothermicity is compensated by a decrease in entropy since the average Gibbs energy of SDS binding is independent of the extent of amidation. At pH 3.2 where the catalase carboxyl groups are largely un-ionized amidation still increase the exothermicity of the interaction with SDS. It is suggested that at low pH the SDS anion interacts favourably with the resonance stabilized O-protonated form of amidated side chains.     

Thermochemical studies on the interaction between sodium n-dodecyl sulphate and catalases

The enthalpies of interaction between bovine catalase and sodium n-dodecyl sulphate (SDS) in aqueous solutions of pH 3.2,6.4 and 10.0 have been measured over a range of SDS concentrations by microcalorimetry at 25°C. The enthalpies increase with decreasing pH and with increasing SDS concentration and largely arise from the interations between the anionic head group of SDS and the cactionic amino acid residues on the protein. Chemically modified catalase in which a proportion of carboxylic acid groups have been coupled with either glycine methyl ester or ethylenediamine have been prepared and characterized in terms of their enzymic activities, spectral properties and sedimentation behaviour. The enthalpies of interaction of these catalases with SDS have been studied at pH 6.4. The results of the experiments suggest that the enthalpies of interaction with SDS can be correlated with the ratio of cationic to anionic amino acid residues on the surface of the catalase molecules and hence the nominal net surface charge. The variation in the enthalpy of interaction of catalases with surface charge, as a consequence of variation in pH, differs from the variation with charge at constant pH possibly due to the thermal effect of proton binding to the catalase—complexes.     

Kinetic approach to the interaction of sodium n-dodecyl sulphate with heme enzymes

The kinetics of interaction of sodium n-dodecyl sulphate (SDS) with catalase has been studied by absorbance and fluorescence changes. The results have been compared with circular dichroism spectra and activity measurements. The tertiary structure of catalase is modified by SDS in the monomeric and micellar form. The secondary structure of catalase is altered only in the presence of SDS micelles. On the other hand, neither spectroscopic properties nor activity of horseradish peroxidase change in the presence of SDS below micellar concentration. In the presence of SDS micelles, however, changes of secondary and tertiary structure of this protein are detected. The reason for relatively high stability of horseradish peroxidase in the presence of SDS is discussed.     

Cooperativity and effects of ionic strength on the binding of sodium n-dodecyl sulphate to lysozyme

The binding of sodium n-dodecyl sulphate to lysozyme has been measured by equilibrium dialysis at 25°C and pH 3.2 over a range of ionic strengts from 0.0119 to 0.2119. Binding isotherms in the region corresponding to ionic binding between the surfactant anions and cationic amino acid residues on the protein have been interpreted in terms of the Hill equation and exhibit positive cooperativity with Hill coefficients in the region of 7–11. The Gibbs energies of binding have been calculated from the Hill binding constants and from the Wyman binding potentials. The stability of the surfactant-protein complexes is discussed in relation to the stability of surfactant micelles. Ionic binding of the surfactant is weakened and hydrophobic binding strengthened by increasing ionic strength.     

Thermodynamic studies on the interaction between sodium n-dodecyl sulphate and histone H2B

The thermodynamic parameters for the interaction of the anionic detergent sodium n-dodecyl sulphate (SDS) with H2B at pH 3.2, 6.4 and 10 have been measured at 27 degrees C and 37 degrees C by equilibrium dialysis to determine the Gibbs energies of detergent binding. The data have been used to obtain the enthalpy of interaction from the temperature dependence of the equilibrium constants from the Van't Hoff relation. The enthalpy of interaction between H2B and SDS is endothermic at pH 3.2, 6.4 and 10. The shapes of the enthalpy curves at pH 3.2 and 10 show some small exothermic contribution which probably indicates folding of H2B. The interactions of H2B-SDS are dominated by the increase in entropy on detergent binding. The larger negative free energy, enthalpy and entropy changes at pH 6.4 are consistent with greater denaturation relative to pH 3.2 and 10.     

Dissociation of bovine and bacterial catalases by sodium n-dodecyl sulfate

The dissociation of beef liver and bacterial (Micrococcus lysodeikticus) catalases by the action of sodium n-dodecyl sulfate (SDS) has been investigated as a function of SDS concetration and time by ultracentrifugation. The rate of dissociation of beef liver catalase is found to be much faster than that for bacterial catalase in 25 mM SDS at pH 7.0. Beef liver catalase is dissociated into its four subunits after 24 h, whereas bacterial catalase is not completely dissociated after 36 days of incubation. The binding of SDS to beef liver catalase obeys a Hill equation with a cooperativity exponent of 2.0 and a binding constant of 440. The initial interaction of SDS with beef liver catalase can be detected by microcalorimetry, whereas the mixing of SDS with bacterial catalase is athermal. Bacterial catalase retains enzymic activity in the presence of SDS, whereas beef liver catalase is completely deactivated at SDS concentrations above 5 mM. Beef liver catalase is more sensitive to acid denaturation than bacterial catalase, and the rate of dissociation for both catalases is sixth-order in proton concentration. Comparison of the amino acid analysis of the two catalases shows that bacterial catalase has a smaller number of lysyl residues and a larger number of glutamyl residues than beef liver catalase. Taken together these structural differences could lead to a reduced affinity of bacterial catalase for the binding of SDS as observed.     

Unusual behaviour of the interaction of sodium n-dodecyl sulphate with calf thymus histone H2B in aqueous solution

The binding of sodium n-dodecyl sulphate (SDS) to calf thymus histone H2B was studied in the pH range 3.2-10 by equilibrium dialysis at 27 and 37 degrees C. The binding data have been used in terms of the Scatchard equation showing unusual plots with minima. No theoretical model gives Scatchard plots with such conditions, except for a combination of two types of binding with large differences in the Hill coefficients and binding affinity, i.e. a combination of negatively and positively cooperative binding sites.     

Suicide-peroxide inactivation of horseradish peroxidase in the presence of sodium n-dodecyl sulphate: a study of the enzyme deactivation kinetics

In the presence of the anionic surfactant sodium n-dodecyl sulphate (SDS), horseradish peroxidase (HRP) undergoes a deactivation process. Suicide inactivation of horseradish peroxidase by hydrogen peroxide(3 mM) was monitored by the absorbance change in product formation in the catalytic reaction cycle. The progress curve of the catalytic reaction cycle was obtained at 27degrees C and phosphate buffer 2.5 mM (pH = 7.0). The corresponding kinetic parameters i.e., intact enzyme activity (alpha i); the apparent rate constant of suicide inactivation by peroxide (ki); and the apparent rate constants of enzyme deactivation by surfactant (kd) were evaluated from the obtained kinetic equations. The experimental data are accounted for by the equations used in this investigation. Addition of SDS to the reaction mixture intensified the inactivation process. The deactivation ability of denaturant could be resolved from the observed inactivation effect of the suicide substrate by applying the proposed model. The results indicate that the deactivation and the inactivation processes are independent of each other.     

Inactivation of glucose oxidase by diperoxovanadate-derived oxidants.

Inactivation of glucose oxidase occurred in the presence of bromide, vanadate, H(2)O(2), and phosphate (the bromide system), and this was prevented by NADH or phenol red, a bromine acceptor. Glucose oxidase present during the reaction between diperoxovanadate and a reduced form of vanadate, vanadyl (the vanadyl system), but not added after mixing the reactants, was inactivated, and this was accompanied by a loss of binding of the dye, Coomassie blue, to the protein. The transient intermediate of the type OVOOV(O(2)), known to form in these reactions and used in the oxidation of bromide ion and NADH, appears to be responsible for inactivating glucose oxidase. In both systems, the inactivation of the enzyme was prevented by histidine and DTT, known to quench singlet-oxygen. By direct measurement of 1270-nm emission of singlet-oxygen, its generation was demonstrated in the bromide system, and in the reaction of hypohalous acids with diperoxovanadate, but not in the vanadyl system. By themselves both hypohalous acids, HOCl and HOBr inactivated glucose oxidase, and their prior reaction with H(2)O(2) during which singlet-oxygen was released, protected the enzyme. The results provide support for possible oxidative inactivation of glucose oxidase by diperoxovanadate-derived oxidants.     

The interaction between Beta-lactoglobulin and sodium N-dodecyl sulphate.

1. The binding of sodium n-dodecyl sulphate to beta-lactoglobulin was studied in the pH range 3.5-7.0 by equilibrium dialysis, ultracentrifugation and microcalorimetry. 2. At low binding concentrations (less than 30 bound surfactants anions per protein molecule) the complexes formed aggregates in solution. 3. At higher binding concentrations aggregation does not occur at low ionic strength (0.01 mol/litre), but continues at high ionic strength (0.1 mol/litre). 4. At 25 degrees C the enthalpy of interaction of sodium n-dodecyl sulphate with beta-lactoglobulin can be interpreted as the sum of the enthalpies of formation of a complex with 2 bound surfactant anions, with an enthalpy change of -9.5 kJ-mol-1 of bound surfactant, and complexes containing at least 22 bound surfactant anions, with limiting enthalpies per bound surfactant anion of -12.4 kJ-mol-1 at pH 3.5 and -3.25 kJ-mol-1 at pH 5.5. 5. The binding of surfactant and the enthalpy of interaction at pH 3.5 ARE NOT SIGNIFICANTLY AFFECTED BY THE ADDITION Of 8 M-urea. 6. The data indicate that at low binding concentrations the interaction is of an ionic nature, and is accompanied by a conformational change in the protein.     

Exploration of pressure-induced dissociation of pyruvate oxidase.

The dissociation of pyruvate oxidase (PO) caused by pressure up to 220 MPa at various conditions was explored by measuring the intrinsic fluorescence spectra and polarization. At 5 degrees C and pH 7.6 the standard volume change (deltaV0) and free energy upon dissociation of the enzyme is -220 ml/mol and 29.83 kCal/mol, respectively. It was found that FAD was irreversibly removed during the pressure-dissociation of the enzyme. A much smaller standard volume change (-153 ml/mol) and lower free energy (24.92 kCal/mol) of apo-pyruvate oxidase (apo-PO) compared with the native enzyme indicated that FAD played very important role in stabilizing the enzyme and significantly influenced the standard volume change. The substrate pyruvic acid can significantly stabilize the enzyme against pressure in spite the standard volume for the enzyme in this case has a big increase relative to the native enzyme. The comparison of the intrinsic fluorescence of the native and the activated enzyme obtained by limited proteolysis indicated that the physical separation of alpha-peptide from the enzyme only occurred when the subunits were dissociated from each other under pressure.     

Enhancement of glutathione levels in mouse peritoneal macrophages by sodium arsenite, cadmium chloride and glucose/glucose oxidase

Glutathione content of mouse peritoneal macrophages markedly increased when they were exposed to insulting agents like sodium arsenite, cadmium chloride, and glucose/glucose oxidase which generates hydrogen peroxide. This increase was attributed to the induction of the cystine transport activity by these agents. The transport activity for other amino acids was not induced, but rather diminished by these agents. Heat shock treatment did not induce the cystine transport activity, nor did it augment glutathione. Since glutathione protects cells against the cytotoxic effects of these agents, the induction of the cystine transport activity constitutes a protective mechanism related to the stress caused by the agents. The protein component(s) for cystine transport may fall into the category of the stress protein.