scholarly journals Binding of branched-chain 2-oxo acids to bovine serum albumin

1982 ◽  
Vol 204 (1) ◽  
pp. 265-272 ◽  
Author(s):  
G Livesey ◽  
P Lund
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Human Plasma ◽  
Bovine Serum ◽  
Equilibrium Dialysis ◽  
Rat Plasma ◽  
Gel Chromatography ◽  
High Concentration ◽  
Branched Chain ◽  
Inhibition Studies

1. Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. 2. Equilibrium-dialysis data suggest a two-side model for binding in Krebs-Henseleit saline at 37 degrees C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2-oxovalerate, k1 = 8.7 × 10(3) M-1, k2 = 0.09 × 10(3) M-1; 3-methyl-2-oxovalerate, k1 = 9.8 × 10(3) M-1, k2 = 0.08 × 10(3) M-1; 3-methyl-2-oxobutyrate, k1 = 1.27 × 10(3) M-1, k2 = less than 0.05 × 10(3) M-1. 3. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4, gave the following thermodynamic parameters: primary site delta H0(1) = -28.6kJ . mol-1 and delta S0(1) = -15.2J . mol-1 . K-1 (delta G0(1) = -24.0kJ . mol-1 at 37 degrees C) and secondary sites delta H0(2) = -25.4kJ . mol-1 and delta S0(2) = -46.1J . mol-1 . K-1 (delta G0(1) = -11.2kJ . mol-1 at 37 degrees C). Thus binding at both sites is temperature-dependent and increases with decreasing temperature. 4. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. 5. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. 6. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.

Screening radioimmunoassay for aldosterone in preheated plasma without extraction and chromatography.

1980 ◽  
Vol 26 (1) ◽  
pp. 41-45
Author(s):  
T M Connolly ◽  
L Tibor ◽  
K H Gless ◽  
P Vecsei
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Binding Proteins ◽  
Equilibrium Dialysis ◽  
Screening Method ◽  
Plasma Aldosterone ◽  
Plasma Samples ◽  
Bovine Serum Albumin Conjugate ◽  
Native Plasma

Abstract We directly estimated plasma aldosterone radioimmunologically with use of an antiserum raised against an aldosterone-3-oxime/bovine serum albumin conjugate, the estimation being on samples with and without heating (60 degrees C), and diluted and undiluted. Values so obtained were compared with those by radioimmunoassay after extraction and chromatography. The correlation--even negative values were obtained--was poorest when the steroid was directly estimated in nonheated, undiluted plasma. Correlations were best (r = 0.918) for preheated and diluted native plasma, and the interassay CV was 9.8% (n = 57). However, there were some extraordinarily high values. After equilibrium dialysis of native and preheated (60 degrees C) plasma (15 plasma samples), the percentages of apparent free aldosterone and cortisol increased from 51.4 +/- 2.6% (SEM) to 64.3 +/- 1.6% and from 11.5 +/- 2.2% to 61.1 +/- 1%, respectively. We conclude that aldosterone-binding proteins play a role in direct radioimmunoassays of aldosterone in plasma, but by heating (with or without diluting) the plasma, direct assay can be used as a simple, fast, and inexpensive screening method.

scholarly journals Effect of arsenic on paracetamol binding to bovine serum albumin using site specific probes

2012 ◽  
Vol 1 (11) ◽  
pp. 361-365 ◽  
Author(s):  
Riaz Uddin ◽  
Nadia Saffoon ◽  
Md. Ashraful Alam
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Equilibrium Dialysis ◽  
Pharmaceutical Journal ◽  
Specific Probe ◽  
Free Concentration ◽  
Site Specific ◽  
Warfarin Sodium ◽  
High Affinity Binding Site

Arsenic contamination in groundwater is a global health challenge. A large number of people worldwide are affected by arsenic poisoning. Paracetamol is a widely used analgesic-antipyretic drug. Effect of arsenic on paracetamol binding to protein has been investigated using two site specific probes and equilibrium dialysis method was used for the experiment. In absence of any site specific probes free concentration of paracetamol bound to bovine serum albumin increased from 3.95 ± 1.164% to 25.36 ± 1.164%. In presence of site-I specific probe warfarin sodium the % release of drug was steady at around 14%. But in presence of site-II specific probe an increment of free drug concentration was observed from 14.38 ± 1.164% to 54.72 ± 1.552%. Thus it can be assumed that the free concentration of paracetamol was increased to a greater extent in presence of arsenic and probably arsenic bound to site-II of BSA. Thus arsenic may displace paracetamol by binding with high affinity binding site, site-II in the BSA and probably arsenic has little effect to site-I.DOI: http://dx.doi.org/10.3329/icpj.v1i11.12061 International Current Pharmaceutical Journal 2012, 1(11): 361-365 

The Partial Purification of Two β-N-Acetyl-D-hexosaminidases from Porcine Kidney

1972 ◽  
Vol 50 (5) ◽  
pp. 563-573 ◽  
Author(s):  
Stephen J. Wetmore ◽  
Jacob A. Verpoorte
Keyword(s):  
Amino Acid ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Gel Chromatography ◽  
Porcine Kidney ◽  
Pig Kidney ◽  
Two Peaks

Two distinct fractions showing both β-N-acetyl-D-glucosaminidase (EC 3.2.1.30) and β-N-acetyl-D-galactosaminidase activity were isolated and purified from pig kidney. These preparations, which were designated A and B, were not stable during gel chromatography or prolonged dialysis. Final purifications of 600-fold for enzyme A and 440-fold for enzyme B were obtained.Gel electrophoresis and ultracentrifugation studies indicated heterogeneity in both preparations. The amino acid compositions of both preparations were very similar. Ultracentrifugation studies suggested the formation of subunits in the presence of 5 M guanidine–HCl and 1 mM dithiothreitol.A study of the enzymatic properties also showed great similarities between the two enzyme forms. Both enzymes had identical Michaelis–Menten constants of 1.88 mM for p-nitrophenyl-β-N-acetyl-D-glucosaminide and 0.38 mM for p-nitrophenyl-β-N-acetyl-D-galactosaminide. Although bovine serum albumin enhanced the activity of the enzymes it did not change the Km values. The pH-rate profiles of both enzymes with the substrate p-nitrophenyl-β-N-acetyl-D-glucosaminide showed two peaks. When p-nitrophenyl-β-N-acetyl-D-galactosaminide was used as substrate, only one peak was observed in the pH–rate profiles. However, in this case a distinct shoulder could be detected in these peaks. Heating at 50° destroyed the activities of both forms of the enzyme rapidly, but addition of bovine serum albumin protected against heat inactivation.

scholarly journals The binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios

1974 ◽  
Vol 137 (3) ◽  
pp. 575-578 ◽  
Author(s):  
Geoffrey Allen
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Sodium Dodecyl Sulphate ◽  
Bovine Serum ◽  
Equilibrium Dialysis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Weight Ratio ◽  
Binding Ratio ◽  
High Binding

The extent of binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios was investigated by gel filtration. The weight ratio of bound sodium dodecyl sulphate to bovine serum albumin increases with the NaCl concentration, and, except at low salt concentrations, with the concentration of sodium dodecyl sulphate. In the presence of 1.0g of sodium dodecyl sulphate/l, the binding ratio varied from 1.0 (at 0.04m-Na+) to 2.2 (at 0.44m-Na+). In the presence of 0.24m-Na+, the binding ratio increased with sodium dodecyl sulphate concentration, from 0.9 (0.2g of sodium dodecyl sulphate/l) to 2.0 (5g of sodium dodecyl sulphate/l), at 26°C, in a dilute sodium phosphate buffer. No significant dependence of the binding ratio upon temperature in the range 26–45°C was observed. These results differ from those of Reynolds & Tanford (1970a) obtained by equilibrium dialysis.

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