A theoretical elucidation of bilirubin interaction with HSA's lysines: first electrostatic binding site in IIA subdomain

The electrostatic interaction of amino acid lysines 190, 195 and 199 of human serum albumin (HSA) with bilirubin have been investigated using molecular dynamic simulations, QM and QM/MM minimization methods. In this study two methodological approaches have been employed. In the first approach X-ray structure and the structure obtained from the molecular dynamic simulation of subdomain IIA of HSA in vacuum have been utilized. Interactions have been evaluated with the segment 186-200 of the cited subdomain. Calculations on the X-ray structure of above segment indicate an effective interaction of the lysine 195 with bilirubin, although that of the lysine 190 is also found considerable in this structure. Performing simulation in vacuum, it has been revealed that except for the lysine 195, the other two lysine residues (190 and 199) could not be considered as centers of interaction. Such finding, which is in accord with experimental data, lends support to the procedure employed in this study. NBO analyses suggest that tasks to achieve a structure indicating bilirubin interaction with the lysine 195 from the 186-200 segment extracted from X-ray structure, results in a structure that lacks any electrostatic interaction. In fact, it has been found that the stability of the latter species can be attributed to the H-bonding interaction of the glutamate 188 with both bilirubin and the lysine 195. Further NBO analysis on the structure of the same species, while achieved after molecular dynamic simulation on subdomain IIA in vacuum has revealed that a favorable electrostatic interaction between the lysine 195 and bilirubin has occurred. Besides, H-bonding interaction of the glutamate 188 with bilirubin has been evident in the same species. For the second approach, presence of water molecules and ions has been considered to simulate condensed medium. Applying docking, conformational sampling, and QM/MM minimization steps in sequence, a structure has been achieved which presents a specific interaction between epsilon-NH3(+) group of the lysine 195 residue and the lactam oxygen atom of bilirubin. NBO analyses suggest that above electrostatic interaction is combined with hydrogen bonding interaction between same two groups. Moreover, a hydrogen bond between oxygen atom of bilirubin's acetate group and alpha-NH group of lysine 195 has been observed. Molecular orbital calculations have been presented which support the NBO analyses.     

Spectrophotometric assay for horseradish peroxidase activity based on pyrocatechol-aniline coupling hydrogen donor

The hydrogen donor couples pyrocatechol-aniline and phenol-aminoantipyrine in the presence of hydrogen peroxide were compared as chromogens for horseradish peroxidase (HRP) assay. UV-Visible spectroscopy and high-performance liquid chromatography analysis indicated that during the HRP biocatalytic process, pyrocatechol-aniline was converted to a pink-colored reagent with a lambda(max) of 510 nm, which was used in the assay of HRP activity. Electrochemical studies revealed adequate electron transfer ability for this color reagent to serve as a proper mediator for HRP also. Using pyrocatechol-aniline a higher sensitivity and lower detection limit was obtained relative to those of the phenol-aminoantipyrine couple, which is commonly used for HRP assay. A relative standard deviation of 2.9% was obtained for 20 HRP activity measurements, indicating a satisfactory reproducibility for this method. In addition, kinetic parameters of K(m) (12.5mM) and V(max) (12.2 mM min(-1)mg(-1)) were calculated for pyrocatechol-aniline. Regarding the superiority of pyrocatechol-aniline, this couple is suggested to be a better hydrogen donor for the HRP spectrophotometric assay.     

Suicide-peroxide inactivation of microperoxidase-11: a kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H2O2. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H2O2] > [AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, alphai, and the apparent inactivation rate constant, ki, were obtained as 0.282 +/- 0.006 min(-1) and 0.497 +/- 0.013(-1) min at [H2O2] = 1.0 mM, 27 degrees C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).     

Relationship between the Structure and Chaperone Activity of Human αA-Crystallin after Its Modification with Diabetes-Associated Oxidative Agents and Protective Role of Antioxidant Compounds

Biochemistry (Moscow) - The study was aimed to evaluate the impact of peroxynitrite (PON, oxidative stress agent in diabetes), methylglyoxal (MGO, diabetes-associated reactive carbonyl compound),...     

Effect of Homocysteine Thiolactone on Structure and Aggregation Propensity of Bovine Pancreatic Insulin

Homocysteine thiolactone (HCTL) is a cyclic thioester of homocysteine, showing high reactivity toward lysine residues of proteins. In the present study the structural properties and aggregation propensity of bovine pancreatic insulin were studied in the presences of increasing concentration of HCTL (0–500 μM), using different spectroscopic techniques. As shown in this study, HCTL induces gross structural alterations and subsequently aggregation of insulin in a dose dependent manner. Also induction of insulin aggregation by HCTL occurs in a sequential process, where native protein with alpha-helical abundant structure gradually transforms into partially folded conformations with the significant amount of beta-sheet. Since C-terminal B-chain of insulin plays a critical role in stability of this protein, the structural alteration/aggregation induced by HCTL can be consequence of homocysteinylation of the only Lysine residue (Lys29) on its B-chain. This study may have important implications regarding the effect of HCTL on structure of insulin particularly in the pathological states linked to hyperhomocysteinemia.     

Effects of calcium binding on the structure and stability of human growth hormone

Thermodynamic analysis of calcium ions binding to human growth hormone (hGH) was done at 27 °C in NaCl solution, 50 mM, using different techniques. The binding isotherm for hGH-Ca²⁺ was obtained by two techniques of ionmetry, using a Ca²⁺-selective membrane electrode, and isothermal titration calorimetry. Results obtained by two ionmetric and calorimetric methods are in good agreement. There is a set of three identical and non-interacting binding sites for calcium ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 52 μM and −17.4 kJ/mol, respectively. Temperature scanning UV–vis spectroscopy was applied to elucidate the effect of Ca²⁺ binding on the protein stability, and circular dichroism (CD) spectroscopy was used to show the structural change of hGH due to the metal ion interaction. Calcium ions binding increase the protein thermal stability by increasing of the alpha helix content as well as decreasing of both beta and random coil structures.     

The Correlation of RNase A Enzymatic Activity with the Changes in the Distance between Nε2-His12 and Nδ1-His119 Upon Addition of Stabilizing and Destabilizing Salts

The effect of stabilizing and destabilizing salts on the catalytic behavior of ribonuclease A (RNase A) was investigated at pH 7.5 and 25°C, using spectrophotometric, viscometric and molecular dynamic methods. The changes in the distance between N_ε2 of His₁₂ and N_δ1 of His₁₁₉ at the catalytic center of RNase A upon the addition of sodium sulfate, sodium hydrogen sulfate and sodium thiocyanate were evaluated by molecular dynamic methods. The compactness and expansion in terms of Stokes radius of RNase A upon the addition of sulfate ions as kosmotropic salts, and thiocyanate ion as a chaotropic salt, were estimated by viscometric measurements. Enzyme activity was measured using cytidine 2′, 3′-cyclic monophosphate as a substrate. The results from the measurements of distances between N_ε2 of His₁₂ and N_δ1 of His₁₁₉ and Stokes radius suggest (i) that the presence of sulfate ions decreases the distance between the catalytic His residues and increases the globular compactness, and (ii) that there is an expansion of the enzyme surface as well as elongation of the catalytic center in the presence of thiocyanate ion. These findings are in agreement with activity measurements.     

Calorimetric and Binding Dissections of HSA Upon Interaction with Bilirubin

The interactions between bilirubin and human serum albumin (HSA) were studied by isothermal titration calorimetry (ITC) and UV–vis spectrophotometry at 27°C in 100 mM phosphate buffer pH 7.4 containing 1 mM EDTA. The biphasic shape of the HSA–bilirubin binding curve depicted the existence of two bilirubin binding sets on the HSA structure which had distinct binding interactions. Each binding set contained one or more bilirubin binding site. The first binding set at subdomain IIA included one binding site and had a more hydrophobic microenvironment than the other two binding sites in the second bilirubin binding set (subdomain IIIA). With our method of analysis, the calculated dissociation constant of the first binding site is 1.28×10⁻⁶ M and 4.80×10⁻⁴ M for the second and third binding sites. Here, the typical Boltzmann’s equation was used with a new approach to calculate the dissociation constants as well as the standard free energy changes for the HSA–bilirubin interactions. Interestingly, our calculations obtained using the Wyman binding potential theory confirmed that our analysis method had been correct (especially for the second binding phase). The molar extinction coefficient determined for the first bound bilirubin molecule depicted that the bilirubin molecules (in low concentrations) should interact with the nonpolar microenvironment of the first high affinity binding site. Binding of the bilirubin molecules to the first binding site was endothermic (ΔH^o>0) and occurred through the large increase in the binding entropy established when the hydrophobic bilirubin molecules escaped from their surrounding polar water molecules and into the hydrophobic medium of the first binding site. On the other hand, the calculated molar extinction coefficient illustrated that the microenvironment of the second binding set (especially for the third binding site) was less hydrophobic than the first one but still more hydrophobic than the buffer medium. The binding of the third bilirubin molecule to the HSA molecule was established more through exothermic (electrostatic) interactions.     

Conformational and Structural Analysis of Bovine β Lactoglobulin-A Upon Interaction with Cr+3

Thermodynamic studies have been made on the effect of Cr⁺³ on the conformation and structure of bovine β lactoglobulin-A, (Blg-A) in 50 mM sodium chloride solution at 27°C using isothermal titration calorimetry (ITC), circular dichroism (CD) and fluorescence spectroscopy. There is a set of six identical and independent binding sites for Cr⁺³ by a dissociation binding constant of 124 μM and the molar enthalpy of binding −17.8 kJ/mol. Circular dichroism studies do not show any significant change in the secondary structure of the protein after the binding of chromium ion on the Blg-A. Fluorescence spectroscopy studies do not show any considerable change in the tertiary structure of Blg-A due to the increasing of Cr⁺³ in low concentration. However, occupation of fourth and fifth binding sites for chromium ions induce partially unfolding in the tertiary structure of the protein resulted from solvent – exposed hydrophobic patches on BLG-A.