Binding of platelet activating factor to albumin

Binding of platelet activating factor (1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine) to albumin is an important facet of the biological activity of this phospholipid. Measurement of that binding has been hampered by the physical nature of the lipid, which made estimation of the free and bound concentrations difficult. With the use of ultracentrifugation to generate an albumin gradient and to produce a region free of protein, the successful measurement of free PAF and PAF bound to albumin was accomplished. This study has demonstrated that PAF binds to albumin at four binding sites and that the average equilibrium dissociation constant for this binding is 1.10(-7) M. Consideration of these data has led to the hypothesis that the receptor active form of PAF is the albumin-PAF complex, rather than free PAF.     

Competition of drugs to serum albumin in combination therapy

The mechanism of cooperative binding of both cytarabine and fluorouracil, used in combination therapy, to the transporting protein [bovine serum albumin (BSA)] has been investigated. Present study shows a strategy of estimating the kind of competition between these drugs with the use of uv and NMR spectroscopy. Two mechanisms of the competition to the transporting protein are proposed. For the quantitative investigations the effect of the protein on both the line width and chemical shifts of the NMR signals of the 5-fluorouracil and cytarabine was analyzed. The pi-pi interaction between the pyrimidine ring of the drugs and the aromatic residues of the protein has been postulated. The binding site for both 5-fluorouracil and cytarabine on BSA was found to be situated in the hydrophobic IIA subdomain. The competition of these two drugs and the removal of 5-fluorouracil by cytarabine from the common binding site in serum albumin tertiary structure are observed.     

Competition of antibacterial drugs for binding sites of human serum albumin

Simultaneous binding of two drugs to human serum albumin (HSA) was studied by flow microcalorimetry. The following drug pairs were used: sulfadimethoxine and cefazolin. Sulfadimethoxine and dicloxacillin, sulfadimethoxine and chlortetracycline. A procedure for estimating the calorimetric titration curves in competing binding of the drugs to the HSA homogeneous active site is described. Comparison of the theoretical and experimental titration curves enabled detection of the ligand competition for the biopolymer binding site. It was shown that sulfadimethoxine displaced cefazolin in the HSA active site, the nature of the HSA association with dicloxacillin and sulfadimethoxine was independent and binding of doxycycline or chlortetracycline to HSA had no influence on sulfadimethoxine interaction with protein.     

Dynamics of human serum albumin studied by acoustic relaxation spectroscopy

Sonic absorption spectra of solutions of human serum albumin (SA) in water and in aqueous phosphate buffer systems have been measured between 0.2 and 2000 MHz at different temperatures (15-35 degrees C), pH values (1.8-12.3), and protein concentrations (1-40 g/L). Several spectra, indicating relaxation processes in the whole frequency range, have been found. The spectra at neutral pH could be fitted well with an analytical function consisting of the asymptotic high frequency absorption and two relaxation contributions, a Debye-type relaxation term with discrete relaxation time and a term with asymmetric continuous distribution of relaxation times. Both relaxation contributions were observed in water and in buffer solutions and increased with protein concentration. The contribution represented by a Debye-type term is practically independent of temperature and was attributed to cooperative conformational changes of the polypeptide chain featuring a relaxation time of about 400 ns. The distribution of the relaxation times corresponding to the second relaxation contribution was characterized by a short time cutoff, between about 0.02 and 0.4 ns depending on temperature, and a long time tail extending to microseconds. Such relaxation behavior was interpreted in terms of solute-solvent interactions reflecting various hydration layers of HSA molecules. At acid and alkaline pH, an additional Debye-type contribution with relaxation time in the range of 30-100 ns exists. It seems to be due to proton transfer reactions of protein side-chain groups. The kinetic and thermodynamic parameters of these processes have been estimated from these first measurements to indicate the potential of acoustic spectra for the investigation of the elementary kinetics of albumin processes.     

Competition of cytarabine and aspirin in binding to serum albumin in multidrug therapy

The aim of this study was to describe a competition between cytarabine (araC) and aspirin (ASA) in binding with bovine serum albumin (BSA). High-affinity binding sites for both drugs were determined using a spectrofluorimetric method. Cytarabine as well as aspirin binds in the IIA hydrophobic subdomain of the transporting protein. Binding constants for araC-BSA and ASA-BSA were calculated by the Scatchard method. Analysis of ultraviolet (UV) difference spectra showed that araC, which has a higher affinity to BSA in comparison to ASA [Ka(araC) > Ka(ASA)] displaces ASA in high-affinity binding sites.The competition between drugs in low-affinity binding sites was investigated using (1)H nuclear magnetic resonance (NMR) and 13C-NMR spectra. We concluded that in the low-affinity binding sites cytarabine decreases the affinity of albumin toward aspirin. However, the interaction between araC and BSA becomes more difficult in the presence of aspirin.     

Tetracycline-Cu(II) photo-induced fragmentation of serum albumin

The protein-damaging potential of photosensitized tetracycline hydrochloride alone and in combination with the metal ion Cu(II) was assessed using serum albumin as a model protein. Exposure of tetracycline to white light in an aqueous solution triggered the generation of significant amounts of reactive oxygen species (ROS) and engendered substantial protein damage. The appearance of distinct low-molecular-mass protein bands on 10% SDS-polyacrylamide gel ascertained the tetracycline concentration-dependent fragmentation of albumin. Photoexcited tetracycline in combination with 100 microM Cu(II) enhanced the protein fragmentation process with concurrent increase in free radical production. The significant release of acid-soluble amino groups and carbonyl groups from treated albumin provided quantitative estimation of protein fragmentation at 0-1.0 mM concentrations of tetracycline. Cu(II) ions per se did not cause any perceptible protein damage. The results with free radical quenchers suggested the role of hydroxyl radicals (*OH) in tetracycline-Cu(II)-induced protein fragmentation, as no superoxide dismutase (SOD)-mediated quenching effect was noted. The generation of free radicals upon tetracycline photoexcitation and consequent protein fragmentation may be considered as important factors in augmentation of tetracycline-induced phototoxic responses.     

Interaction between rat serum phosphorylcholine binding protein and platelet activating factor

The binding of rat serum phosphorylcholine binding protein (PCBP) to platelet activating factor (PAF) has been demonstrated using a HPLC-gel filtration technique. The bulk of the bound [3H]-PAF eluted with a higher molecular weight species of PCBP, possibly an aggregated form of PCBP. A smaller amount of [3H]-PAF co-eluted with the major monomeric species of PCBP. Formation of the PCBP-PAF complex was calcium dependent and could be inhibited by phosphorylcholine, suggesting the involvement of the phosphorylcholine binding site on PCBP. Binding of albumin and alpha 1-acid glycoprotein to PAF was not affected by phosphorylcholine or calcium. The specificity of this binding may explain the inhibitory effect of PCBP and related phosphorylcholine binding proteins on PAF induced aggregation of platelets.     

Dynamics of heme complexed with human serum albumin: a theoretical approach

Human serum albumin (HSA) is the most abundant protein in the blood serum. It binds several ligands and has an especially strong affinity for heme, hence becoming a natural candidate for oxygen transport. In order to analyze the interaction of HSA-heme, molecular dynamics simulations of HSA with bound heme were performed. Based on the results of X-ray diffraction, the binding site of the heme, localized in subdomain IB, was considered. We analyzed the fluctuations and their correlations along trajectories to detect collective motions. The role of H bonds and salt bridges in the stabilization of heme in its pocket was also investigated. Complementarily, the localization of water molecules in the hydrophobic pocket and the interaction with heme were discussed.     

Suppression of lymphokine production: II. Macrophage-dependent inhibition of production of macrophage activating factor

The ability of different populations of macrophages to affect the production of macrophage activating factor (MAF) by stimulated T lymphocytes was investigated. We found that activated macrophages, infiltrating MSV-induced regressing tumors or macrophages recovered from the peritoneum of mice injected with Corynebacterium parvum, were able to actively suppress the production of MAF. MAF production by antigen-stimulated MSV-immune or -alloimmune spleen cells and by normal spleen cells stimulated by Con A was susceptible to macrophage-dependent suppression to a similar extent. In contrast, resident macrophages or those elicited by light mineral oil or proteose-peptone did not affect MAF production. While suppressor macrophages added at the time of the lymphocyte stimulation inhibited MAF production, the same cells added 4–6 hr after stimulation were ineffective. Therefore, it seems that the macrophages suppressed the early events of lymphocyte activation leading to MAF production. Suppressor macrophages, by inhibiting MAF production, may limit the expansion of the cytotoxic activity. This regulation of macrophage functions, mediated by the effects of suppressor macrophages on T lymphocytes, could be responsible for an insufficient antitumor cytotoxic response by macrophages.