ABSTRACT
Lipoxygenases comprise a family of non-heme iron-containing enzymes that catalyze the stereospecific dioxygenation of polyunsaturated fatty acids with 1, 4-cis-cis-pentadiene structure. Hemoglobin, a heme iron-containing protein has been reported to have lipoxygenase activity but the assay conditions that could enhance the activity remain obscure. Therefore, establishment of optimum assay conditions for lipoxygenase activity of hemoglobin could allow modeling of hemoglobin as lipoxygenase. Hemoglobin was extracted from blood of an identified individual of genotype AA. The hemoglobin was dialyzed at 4 degrees C for 24 h against 50 mM Tris-HCl buffers (pH 8.5 and 7.2) and effects of sodium dodecyl sulphate (SDS) and linoleic studied at pH 5.0 and 7.2 with UV-VIS Titration Spectrophotometry. The results show that 3.3, 8.6 and 88.1% concentrations of met-hemoglobin were found in presence of 0.0 mM SDS at pH 5.0 and 7.2, 1.043 mM SDS at pH 7.2 and 0.404 mM SDS at pH 5.0 respectively. Also, the difference spectra of hemoglobin in presence of linoleic acid showed positive peak at 285 nm which suggest the presence of oxodienes--a reaction product of hydroperoxidase activity of lipoxygenase. Formation of met-hemoglobin/met-myoglobin is highly correlated with lipid oxidation. Since highest concentration of met-hemoglobin (88.1%) was observed in presence of 0.404 mM SDS at pH 5.0, lipoxygenase activity of hemoglobin was enhanced in presence of SDS under these conditions.
Subject(s)
Detergents/pharmacology , Hemoglobins/drug effects , Lipoxygenase/drug effects , Sodium Dodecyl Sulfate/pharmacology , Hemoglobins/metabolism , Humans , Linoleic Acid/metabolism , Lipoxygenase/metabolism , Methemoglobin/metabolism , Osmolar Concentration , Oxidation-Reduction , Oxyhemoglobins/metabolism , Spectrophotometry, UltravioletABSTRACT
The interaction of n-dodecyl trimethylammonium bromide (DTAB) with oxyhemoglobin A and oxyhemoglobin S is investigated using UV-visible absorption spectra and chemometric resolution techniques. Oxyhemoglobins (A and S) induced to partial oxidized form (ferrihemoglobin) by DTAB and finally transform to fully oxidized hemichrome. Hemichrome mole fractions of HbS are more than HbA because of more hydrophobic interaction of DTAB-HbS in second set of binding site relative to DTAB-HbA. The visible spectra between 500 and 650 nm are used for identifying the present components in solution because each species of hemoglobin has a specific spectrum in this region. The number of components and mole fraction of mentioned species were determined by employing chemometric resolution techniques. Subspace comparison was used for determination of the number of components in each concentration of hemoglobin and DTAB. After the determination of components, multivariate curve resolution-alternating least square (MCR-ALS) by initial estimates of spectral profiles and proper constraints, was used to resolve the data matrix into pure concentration and spectral profiles. The results show that both number and mole fraction of components which were formed during hemoglobin (HbA and HbS) oxidation by DTAB were initial hemoglobin concentrations independent. Furthermore, in average the mole fraction of hemichrome of HbS is 14.4% more than HbA. On the other hand, the mole fraction of HbA ferrihemoglobin is 15.6% higher than HbS averagely.
Subject(s)
Hemoglobins/chemistry , Quaternary Ammonium Compounds/chemistry , Isomerism , Multivariate Analysis , Spectrophotometry, UltravioletABSTRACT
The kinetics of thermal dissociation of superoxide dismutase (SOD) was studied in 0.05 M Tris-HCl buffer at pH 7.4 containing 10(-4) M EDTA. The number of conformational locks and contact areas and amino acid residues of dimers of SOD were obtained by kinetic analysis and biochemical calculation. The cleavage bonds between dimers of SOD during thermal dissociation and type of interactions between specific amino acid residues were also simulated. Two identical contact areas between two subunits were identified. Cleavage of these contact areas resulted in dissociation of the subunits, with destruction of the active centers, and thus, lost of activity. It is suggested that the contact areas interact with active centers by conformational changes involving secondary structural elements.
Subject(s)
Protein Conformation , Superoxide Dismutase/chemistry , Temperature , Animals , Binding Sites , Cattle , Dimerization , Enzyme Stability , Models, Molecular , Protein Subunits/chemistry , Protein Subunits/metabolism , Superoxide Dismutase/metabolismABSTRACT
The kinetics and thermodynamics of the effects of paraoxon (POX) and ethylparathion (EPA) on choline oxidase (ChOx) were studied. Lineweaver-Burk plots of initial velocity data showed a parallel pattern indicating uncompetitive inhibition versus choline. The inhibition constant (K(I)) obtained from the secondary plots for POX and EPA were 0.14+/-0.01 and 0.48+/-0.05 mM, respectively, suggesting that POX is a more potent inhibitor of ChOx than EPA. UV absorption was used to monitor the denaturation of ChOx by POX and EPA. A decrease in FAD fluorescence associated with the interaction of POX and EPA with ChOx suggested a tertiary structural change. Interaction of the enzyme molecule with POX or EPA resulted in inhibition and subsequently denaturation of the enzyme. The results indicate that inhibition and denaturation of the enzyme by POX and EPA are linked, but not parallel events, with inhibition occurring at lower concentrations with respect to denaturation. This suggests that the loss of initial velocity of the enzyme is an active site specific effect and not due to global conformational changes induced by the inhibitors.
