Nitrosylated bovine serum albumin derivatives as pharmacologically active nitric oxide congeners.
Although nitrosothiols have been suggested to act as regulators of cell (patho)physiology, little is known about the pharmacology of nitrosylated proteins as nitric oxide (NO.) congeners. We describe the molecular consequences of nitrosylating bovine serum albumin (BSA) at multiple specific sites and demonstrate that the product S-nitrosoproteins exert NO.-like activity. The content of nucleophilic nitrosylation sites (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 then selectively positioned on either the endogenous sulfhydryl groups of reduced BSA or the homocysteine moieties of thiolated BSA, respectively. Each resulting S-nitrosoprotein adduct was an oligomeric mixture across the >2000 kDa to approximately 66 kDa molecular mass range. The BSA-derived S-nitrosoproteins were immunoreactive with antibodies against native BSA but evidenced compromised long-chain fatty acid binding. Both types of BSA-derived S-nitrosoproteins suppressed human coronary artery smooth muscle cell proliferation to a similar degree (IC50 approximately 70 microM NO. equivalents) and were significantly more effective antiproliferative agents than a standard NO. donor, DETA NONOate. Antiproliferative bioactivity reflected the NO functionalities carried by each protein, but was independent of molecular mass of the nitrosylated BSA adducts. These data exemplify the rational design and characterization of protein-based S-nitrosothiols as NO. congeners and suggest that such agents could have therapeutic potential as NO delivery systems.
Ewing
J F
JF
NitroMed, Inc., Bedford, Massachusetts 01730, USA.
Young
D V
DV
Janero
D R
DR
Garvey
D S
DS
Grinnell
T A
TA
D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002850:Chromatography, Gel; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D009603:Nitroso Compounds / Q000032:analysis / Q000494:pharmacology*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000494:pharmacology*
D005227:Fatty Acids; D009603:Nitroso Compounds; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide