EFFECT OF NITRIC-OXIDE ON ALBUMIN PALMITATE BINDING

Bovine serum albumin (albumin) was modified by treatment with nitric o xide (NO) to form S-nitrosoalbumin. Analysis of the reduced sulfhydryl groups showed that more than 99% of the albumin was converted to S-ni trosoalbumin. Using a 1:1 molar ratio of protein:palmitate, the unboun d palmitate fraction in the presence of S-nitrosoalbumin was determine d 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 [H-3]palmitic acid. The equilibri um association constants (K-alpha) for albumin-palmitate and S-nitroso albumin-palmitate complexes were determined using the stepwise equilib rium model. The K-alpha for the first and second palmitate binding sit es were (4.6 +/- 1.2) x 10(8) M(-1) and (3.3 +/- 0.5) x 10(7) M(-1) an d (3.1 +/- 0.9) x 10(8) M(-1) and (1.3 +/-:0.8) x 10(8) M(-1) for albu min and S-nitrosoalbumin, respectively. Thus, the increased unbound fr action of palmitate in the presence of S-nitrosoalbumin was apparently due to a decreased binding affinity at the first high-affinity bindin g site. Palmitate uptake by hepatocyte suspensions was 27% higher in t he presence of S-nitrosoalbumin as compared with albumin. This increas e paralleled the increased unbound palmitate fraction. When the albumi n concentration was adjusted to account for the increased unbound frac tion, there was no difference in the palmitate uptake rates between al bumin and S-nitrosoalbumin, Our findings indicate that under condition s where NO concentrations are high (e.g. cirrhosis) and extensive S-ni trosylation of serum albumin occurs, the decreased ligand binding abil ity of S-nitrosoalbumin may be an important consideration when modelin g drug uptake in pathological states.