Complexes of Cu2+ with pyridoxal-5'-phosphate and human and bovine serum albumin

The copper ion Cu2+ bound to serum albumin in the most strong center stabilizes aldimine bonds formed by PLP with epsilon-NH2 group of 4-Lys and alpha-NH2 1-Asp. The stoichometric ratio of the ternary albumin-PLP-Cu2+ complex is 1:2:1. The imidazole rings of histidine residues are involved in binding of copper ions in the first, second, third centers of the albumin molecule. In this case copper ions increase the binding of PLP with the protein stabilizing Schiff bases produced by epsilon-NH2 group of lysine and PLP. The cooper ion bound to serum albumin in the most strong center forms two types of complexes: with rhombic environment in neutral and alkaline media and axial one at pH less than 5,0. On formation of the ternary complex with PLP the rhombic environment is changed to axial.     

Interaction of isofraxidin with human serum albumin

This study was designed to examine the interaction of isofraxidin with human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 3.3 x 10(-6) mol L(-1)-3.0x10(-5) mol L(-1) and HSA concentration at 1.5 x 10(-6) mol L(-1). Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy were used to determine the drug-binding mode, the binding constant and the protein structure changes in the presence of isofraxidin in aqueous solution. Spectroscopic evidence showed that the interaction results in one type of isofraxidin-HSA complex with binding constants of 4.1266 x 10(5) L mol(-1), 3.8612 x 10(5) L mol(-1), 3.5063 x 10(5) L mol(-1), 3.1241 x 10(5) L mol(-1) at 296 K, 303 K, 310 K, 318 K, respectively. The thermodynamic parameters, enthalpy change (DeltaH) and entropy change (DeltaS) were calculated to be -10.08 kJ mol(-1) and 73.57 J mol(-1) K(-1) according to van't Hoff equation, which indicated that hydrophobic interaction played a main role in the binding of isofraxidin to HSA. The experiment results are nearly in accordance with the calculation results obtained by Silicon Graphics Ocatane2 workstation.     

Interaction of taxol with human serum albumin

Taxol (paclitaxel) is an anticancer drug, which interacts with microtuble proteins, in a manner that catalyzes their formation from tubulin and stabilizes the resulting structures (Nogales et al., Nature 375 (1995) 424-427). This study was designed to examine the interaction of taxol with human serum albumin (HSA) in aqueous solution at physiological pH with drug concentrations of 0.0001-0.1 mM, and HSA (fatty acid free) concentration of 2% w/v. Gel electrophoresis, absorption spectra and Fourier transform infrared (FTIR) spectroscopy with self-deconvolution and second-derivative resolution enhancement were used to determine the drug binding mode, binding constant and the protein secondary structure in the presence of taxol in aqueous solution. Spectroscopic evidence showed that taxol-protein interaction results into two types of drug-HSA complexes with overall binding constant of K=1.43 x 10(4) M(-1). The molar ratios of complexes were of taxol/HSA 30/1 (30 mM taxol) and 90/1 (90 mM taxol) with the complex ratios of 1.9 and 3.4 drug molecules per HSA molecule, respectively. The taxol binding results in major protein secondary structural changes from that of the alpha-helix 55 to 45% and beta-sheet 22 to 26%, beta-anti 12 to 15% and turn 11 to 16%, in the taxol-HSA complexes. The observed spectral changes indicate a partial unfolding of the protein structure, in the presence of taxol in aqueous solution.     

Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants

Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

Interaction of human serum albumin with luminol]

Chemoluminescent and fluorescent studies of the interaction between serum albumine and luminole have been carried out. An increase of chemoluminescent intensity and quenching of luminole fluorescence dependent on protein concentration has been observed. Possibility of complex formation and the mechanism of fluorescence quenching with luminole are discussed.     

Interaction of perfluorooctanoic acid with human serum albumin

Background  

Recently, perfluorooctanoic acid (PFOA) has become a significant issue in many aspects of environmental ecology, toxicology, pathology and life sciences because it may have serious effects on the endocrine, immune and nervous systems and can lead to embryonic deformities and other diseases. Human serum albumin (HSA) is the major protein component of blood plasma and is called a multifunctional plasma carrier protein because of its ability to bind an unusually broad spectrum of ligands.     

Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.     

Interaction of divalent metal ions with human serum albumin

ESR study was carried out of the interaction between Zn2+, Cu2+, Ca2+, Mg2+ ions and human serum albumin (HSA) in the presence of Mn2+ ions which depends on pH. Competitive binding of these ions with "manganese-binding" sites of albumin was shown to depend on pH. An analysis of concentration dependence of binding these ions with human serum albumin confirmed earlier supposition about the nature of the binding sites of Mn2+ ions with HSA.     

Physico-chemical properties of ribonuclease A modified with pyridoxal-5'-phosphate]

The physico-chemical properties have been studied of RNase A selectively modified at the E-NH2-group of Lys-7 and Lys-41 with pyridoxal-P. Modification did not affect conformational stability of the protein globule, thus all changes in the molecule of the modified RNase A were localised around the alkylated Lys residue. In the both cases pyridoxyl-P. The residue was shown to be localized in the active site region of the (P-Pxy)-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. (P-Pxy) E-Lys-7-RNase A and its chromophore parts was highly exposed to the solvent. In the Lys-41 derivative, pyridoxamine-P was situated exactly in the active site and is partially hidden in the protein grobule. The pH-dependence of absorption spectra indicates that the chromophore of pyridoxyl-P in modified proteins is quite sensible to the ionic state of its surrounding. The usefulness of pyridoxyl-P as a reporter group was proved in the study with (P-Pxy)-Lys-7-RNase A. Some conformational changes involving His-119 were shown to take place in the course of the enzyme-nucleotide complex formation.     

Interaction of VO ion with human serum transferrin and albumin

The complexation of VO²⁺ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO²⁺ ion occupies three different binding sites, A, B₁ and B₂, distinguishable in the X-band anisotropic spectrum recorded in D₂O. With albumin the results show that a dinuclear complex (VO)₂^dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO²⁺, the multinuclear complex (VO)_x^mHSA is the prevalent species, where x = 5-6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the ⁵¹V A_z values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO²⁺/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO²⁺ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems.     

Interaction of anesthetic supplement thiopental with human serum albumin

Thiopental (TPL) is a commonly used barbiturate anesthetic. Its binding with human serum albumin (HSA) was studied to explore the anesthetic-induced protein dysfunction. The basic binding interaction was studied by UV-absorption and fluorescence spectroscopy. An increase in the binding affinity (K) and in the number of binding sites (n) with the increasing albumin concentration was observed. The interaction was conformation-dependent and the highest for the F isomer of HSA, which implicates its slow elimination. The mode of binding was characterized using various thermodynamic parameters. Domain II of HSA was found to possess a high affinity binding site for TPL. The effect of micro-metal ions on the binding affinity was also investigated. The molecular distance, r, between donor (HSA) and acceptor (TPL) was estimated by fluorescence resonance energy transfer (FRET). Correlation between the stability of the TPL-N and TPL-F complexes and drug distribution is discussed. The structural changes in the protein investigated by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy reflect perturbation of the albumin molecule and provide an explanation for the heterogeneity of action of this anesthetic.     

Interaction of prostaglandin E2 with human serum albumin

Using equilibrium dialysis, protein fluorescence and fluorescent probing as well as chemical modification, the interaction of prostaglandin E2 with human serum albumin was studied. The serum albumin molecule has a highly specific prostaglandin E2-binding site. The binding of prostaglandin causes conformational rearrangements in the protein molecule. The amino group of serum albumin is involved in the interaction with prostaglandin E2. Prolonged exposure of prostaglandin E2 to serum albumin causes partial irreversible binding of prostaglandin molecules to the protein.     

Localization of pyridoxal-5-phosphate, covalently bound with human hemoglobin. Spectrofluorimetric studies]

Human apohemoglobin tryptophan residues were localized in the regions of the protein globule with restricted mobility. By the method of dynamic quenching of phosphopyridoxyl chromophore fluorescence, the heterogeneity of pyridoxal-5-phosphate molecules covalently bound to the human hemoglobin molecules was determined from the accessibility to solvent. The first four pyridoxal-5-phosphate molecules are localized in the hydrophobic regions of the hemoglobin molecule; at the same time, they have a high mobility. One of these molecules is situated at the site inaccessible to the solvent, which coincides with the anion-binding center of the oxyhemoglobin molecule. The next pyridoxal-5-phosphate molecules modify the surface amino groups of the protein. In the apohemoglobin molecule, the pyridoxal-5-phosphate binding sites are more exposed to the solvent, as compared to hemoglobin. In the hemoglobin molecule modified by pyridoxal-5-phosphate, an effective electron excitation energy transfer from tryptophan residues to phosphopyridoxyl chromophores occurs. The effective distances between tryptophanyls of single subunits of hemoglobin and the covalently bound pyridoxal-5-phosphate molecule were estimated to be 19 A for the alpha-subunit and 17 A for the beta-subunit.