THE INTERACTION OF ETHACRYNIC ACID, DIHYDROETHACRYNIC ACID AND CERTAIN THIOL ADDUCTS OF ETHACRYNIC ACID WITH BOVINE SERUM ALBUMIN

¹ Department of Pharmacology and Therapeutics, Medical College of Ohio, Toledo, Ohio

The interaction of bovine serum albumin (BSA) with ethacrynic acid (EA) and certain EA analogs was studied with special emphasis on the role of the BSA thiol group in the binding process. EA was incubated with BSA in mole ratios ranging from 1:1 to 10:1 (EA/BSA) for 15 minutes at 37°C followed by the addition of Ellman's reagent. Only a small reduction in the thiol content of BSA was observed with mole ratios of 1:1 and 2:1 (EA/BSA); however, a substantial (slowly reversible) diminution of the thiol content of BSA occurred when the mole ratio was increased to 5:1 or 10:1. The data support the notion that EA reduces the thiol content of BSA via a direct alkylation of this group. Dihydroethacrynic acid did not reduce the thiol content of BSA when the mole ratios were 2:1 or 5:1 (dihydroethacrynic acid/BSA). Studies in which four relatively stable thiol adducts of EA (EA-2,5-dichlorobenzenethiol. EA-thiophenol, EA-N-acetylmercaptoethylamine and EA-3-mercaptopropionic acid were incubated with BSA indicate that BSA catalyzes, to varying degrees, the release of EA and the accompanying thiol from these adducts. This latter finding may explain: 1) why EA-2,5-dichlorobenzenethiol (3.3 µmol/kg i.v.) and EA-thiophenol (17.0 µmol/ kg i.v.) produce a small but significant diuresis in dogs, and 2) why the most stable of the four thiol adducts of EA, EA-3-mercaptopropionic acid (17.0 µmol/kg, i.v.), is ineffective in normal dogs but elicits a modest diuretic response in dogs in which its body half-time has been prolonged by ligation of the cystic and common bile ducts.