NITROSYLATED BOVINE SERUM-ALBUMIN DERIVATIVES AS PHARMACOLOGICALLY ACTIVE NITRIC-OXIDE CONGENERS

Although nitrosothiols have been suggested to act as regulators of cel l (patho)physiology, little is known about the pharmacology of nitrosy lated proteins as nitric oxide (NO.) congeners. We describe the molecu lar consequences of nitrosylating bovine serum albumin (BSA) at multip le specific sites and demonstrate that the product S-nitrosoproteins e xert NO.-like activity. The content of nucleophilic nitrosylation site s (i.e., free sulfhydryl groups) in native BSA was increased by either reduction with dithiothreitol or thiolation with N-acetylhomocysteine . Fourteen moles of nitrogen monoxide (NO)/mol BSA equivalent were the n selectively positioned on either the endogenous sulfhydryl groups of reduced BSA or the homocysteine moieties of thiolated BSA, respective ly. Each resulting S-nitrosoprotein adduct was an oligomeric mixture a cross the >2000 kDa to approximate to 66 kDa molecular mass range. The BSA-derived S-nitrosoproteins were immunoreactive with antibodies aga inst native BSA but evidenced compromised long-chain fatty acid bindin g. Both types of BSA-derived S-nitrosoproteins suppressed human corona ry artery smooth muscle cell proliferation to a similar degree (IC50 a pproximate to 70 mu M NO. equivalents) and were significantly more eff ective antiproliferative agents than a standard NO. donor, DETA NONOat e. Antiproliferative bioactivity reflected the NO functionalities carr ied by each protein, but was independent of molecular mass of the nitr osylated BSA adducts. These data exemplify the rational design and cha racterization of protein-based S-nitrosothiols as NO. congeners and su ggest that such agents could have therapeutic potential as NO delivery systems.