Effect of nitric oxide on albumin-palmitate binding.
Bovine serum albumin (albumin) was modified by treatment with nitric oxide (NO) to form S-nitrosoalbumin. Analysis of the reduced sulfhydryl groups showed that more than 99% of the albumin was converted to S-nitrosoalbumin. Using a 1:1 molar ratio of protein:palmitate, the unbound palmitate fraction in the presence of S-nitrosoalbumin was determined to be greater (28%) than in the presence of albumin as determined by heptane: water partitioning. NO degradation products neither affected the palmitate heptane:water partition ratio in the absence of binding protein nor the hepatocyte uptake of [3H]palmitic acid. The equilibrium association constants (Ka) for albumin-palmitate and S-nitrosoalbumin-palmitate complexes were determined using the stepwise equilibrium model. The Ka for the first and second palmitate binding sites were (4.6 +/- 1.2) x 10(8) M-1 and (3.3 +/- 0.5) x 10(7) M-1 and (3.1 +/- 0.9) x 10(8) M-1 and (1.3 +/- 0.8) x 10(8) M-1 for albumin and S-nitrosoalbumin, respectively. Thus, the increased unbound fraction of palmitate in the presence of S-nitrosoalbumin was apparently due to a decreased binding affinity at the first high-affinity binding site. Palmitate uptake by hepatocyte suspensions was 27% higher in the presence of S-nitrosoalbumin as compared with albumin. This increase paralleled the increased unbound palmitate fraction. When the albumin concentration was adjusted to account for the increased unbound fraction, there was no difference in the palmitate uptake rates between albumin and S-nitrosoalbumin. Our findings indicate that under conditions where NO concentrations are high (e.g. cirrhosis) and extensive S-nitrosylation of serum albumin occurs, the decreased ligand binding ability of S-nitrosoalbumin may be an important consideration when modeling drug uptake in pathological states.
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, Winnipeg, Canada.
F J
FJ
Burczynski
G Q
GQ
Wang
M
M
Hnatowich
D000818:Animals; D005260:Female; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D009603:Nitroso Compounds; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism* / Q000493:pharmacokinetics; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D012710:Serum Albumin, Bovine / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium
D009603:Nitroso Compounds; D010169:Palmitic Acids; C091665:S-nitrosoalbumin; D014316:Tritium; D012710:Serum Albumin, Bovine; D019308:Palmitic Acid; D009569:Nitric Oxide