Binding Sites Of Bovine Serum Albumin Research Articles

Inhibition of in vitro fertilization of mouse eggs: 3‐quinuclidinyl benzilate specifically blocks penetration of zonae pellucidae by mouse spermatozoa

The fertilization in vitro of mouse with intact zonae pellucidae by mouse cauda epididymal spermatozoa was inhibited in a concentration- dependent fashion by 3-quinuclidinyl benzilate (QNB), normally used as a specific antagonist for the muscarinic class of cholinergic receptors. Inhibition was observed with both cumulus-intact and cumulus-free preparations. QNB at 50 microM inhibited fertilization of cumulus-free eggs by greater than 90% but had no effect on the fertilization of zona-free eggs. At this concentrations, QNB had no adverse effect on sperm motility, nor did it prevent binding of spermatozoa to the zona pellucida. The inhibitory effects of QNB were fully reversible. QNB is therefore a useful specific inhibitor of zona penetration. Spermatozoa in the in vitro fertilization medium bound QNB with a concentration dependence which matched that of the inhibition of fertilization. This binding was saturable and corresponded to 700 pmole/10(7) cells with KD = 10 microM. The in vitro fertilization medium contains 2% (w/v) bovine serum albumin (BSA) which also binds QNB according to the mass action law. The large amount of QNB bound to sperm in this medium appears to be QNB binding to BSA adsorbed on the sperm cell surface: these spermatozoa bind QNB specifically in the absence of BSA with a saturable capacity of only 70 fmole/10(7) cells with KD = 5 nM. Calculation of the distribution of QNB between BSA binding sites and sperm surface binding sites in the in vitro fertilization medium indicates that the specific sperm sites become saturated with the same concentration dependence as inhibition of fertilization. However, the dissociation rate of QNB from sperm in both the presence and absence of BSA is too rapid to permit confirmation of these sites as the locus of the inhibitory effect; this locus remains to be clarified.

Kinetic Studies of the Interaction of Bromphenol Blue with Bovine Serum Albumin by Pressure-jump Method

Abstract The interaction of Bromphenol Blue (BPB) with bovine serum albumin (BSA) was studied statically, by spectrophotometry, and kinetically, by the pressure-jump method. The absorbance changes of BPB were monitored at 20 °C, pH 7.00 in 0.2 M phosphate buffer. The static measurements showed that there were two binding classes. The number of binding sites and the binding constants for each class are n1=1, K1=1.4×107 M−1 and n2=3, K2=9.5×104 M−1. Kinetically, the binding of BPB to the primary binding site of BSA proceeds via at least 4 steps. Two models are offered for the possible binding mechanism. In these models, a fast, probably diffusion controlled, second order step is followed by three first order steps. A correlation between the number of binding steps and the magnitude of the binding constant is discussed. The positive activation entropies associated with the backward reactions of the second and third steps show that these reactions proceed through disordered activation states. From the comparison of the present results to those for other ligands, it was found that the ligand is intimately involved in which activated configuration is adopted.

Drug interactions. VII. The fatty acid binding properties of bovine serum albumin.

The interrelationships between fatty acid and probe binding to bovine serum albumin (BSA) were investigated by a fluorescence method, using 1-anilinonaphthalene-8-sulfonate (ANS) and N-phenyl-1-naphthylamine (NPN) as fluorescent probes. The fatty acidinduced quenching of probe fluorescence is probably due to the displacement of probes from BSA binding sites. It appears that NPN and ANS are bound to BSA at the same sites. Various molar ratios of capric acid, lauric acid and palmitic acid were added to defatted BSA in the presence of ANS, and the association constants of the fatty acids were calculated. Measurements of ANS fluorescence (λmax 465nm) and the fluorescence (λmax 340nm) of the ANS-BSA complex as well as the circular dichroism spectra of BSA in the presence of fatty acids in pH 7.4 phosphate buffer indicated that fatty acids induce different conformational states of the albumin molecule. One of the strongest binding sites of long-chain fatty acids may be the site which is labeled by TNBS (2, 4, 6-trinitrobenzenesulfonic acid), located in the carboxyl-terminal half of the molecule, which does not contain tryptophan residues. It is suggested that the mechanism of inhibition of the probe-BSA binding by capric acid may involve competition for the same binding sites. In contrast, the decreases in probe binding with BSA in the presence of lauric acid. palmitic acid or stearic acid are due not only to a displacement of the probes by fatty acids from the probebinding sites but also to a simultaneous conformational change of the probe-binding sites caused by the interaction of the probes and fatty acids.

Effect of salicylate on the binding of sulfonamides to bovine serum albumin.

Displacement of eight sulfonamides by sodium salicylate from the binding sites of bovine serum albumin (BSA) was studied by the equilibrium dialysis method at pH 7.4 and 37°. Amount of sulfonamides displaced by the addition of salicylate (1 or 3 mM) were linearly correlated with their amount bound to the binding sites of BSA in the absence of salicylate. Double-reciprocal plots of the binding of sulfamethoxazole to BSA with and without salicylate, following Langmuir's isotherms, resulted in linear curves with a common ordinate intercept. This result suggests that the inhibitory effects of salicylate on the binding of sulfamethoxazole to albumin were competitively performed at the same binding site on the protein.

The incorporation of elaidate, oleate and straight-chain saturated fatty acids by mycoplasma strain Y.

Summary: Mycoplasma strain Y grew well in a partly defined medium containing charcoal-treated bovine serum albumin (BSA) with added elaidate, elaidate plus oleate, or elaidate plus a saturated fatty acid containing 10 to 15, 22 or 24 carbon atoms. With elaidate plus a saturated acid containing 16 to 20 carbon atoms there was good growth after a variable lag period. When elaidate was the only fatty acid supplied it comprised from 93 to 95% of the total fatty acids in stationary-phase mycoplasmas, the remainder being derived from contaminating fatty acids in the medium. There was no isomerization of elaidate to oleate. When grown with elaidate plus a shorter-chain saturated acid (C10, C12) or with a long-chain acid (C22, C24), elaidate was incorporated preferentially during the early stages of growth, increasing proportions of the saturated acids being incorporated during the later stages. When grown with elaidate plus a saturated acid of intermediate chain length (C14 to C16), or with elaidate plus oleate, both fatty acids were incorporated in approximately equal proportions throughout growth. The preferential incorporation of elaidate in the early growth stages with some pairs of fatty acids is explained by a competition between the two fatty acids for the BSA binding sites.