Bo Ekman

Binding of drugs to human serum albumin—XV: Characterization and identification of the binding sites of indomethacin

Abstract The binding of indomethacin to human serum albumin (HSA)§ has been studied with HSA immobilized in microparticles of polyacrylamide, by equilibrim dialysis and by circular dichroism. Indomethacin is bound to three sites with high affinity. The association constants are 1.5–2.1 × 10 6 , 3.5 × 10 5 and 5.5 × 10 4 M −1 , respectively, as found by spectropolarimetric titration and from Scatchard plots of data obtained from the studies with microparticles. The evaluation of the CD-data is simplified by the different extrinsic Cotton effects engendered when indomethacin binds to the different sites. With specific probes, binding exclusively to single sites, we have shown that indomethacin primarily binds to the warfarin-(azapropazone-) site and tertiarily to the diazepam-(flurbiprofen-) site on HSA. The identity of the secondary site has not been established. Probenecid does not affect the first site of indomethacin, but improves the binding of indomethacin to the second site, probably via allosteric effects from the third site.

The analytical use of lactate dehydrogenase entrapped in microparticles

Abstract Lactate dehydrogenase (LDH) was immobilized in microparticles of polyacrylamide. The particles were prepared by polymerization of acrylamide and bisacrylamide in an emulsion of water in toluene-chloroform. The polyacrylamide gel formed is macroporous and allows rapid equilibrium between the particles and the environment to take place. The enzyme was shown to be active by reducing pyruvate. The LDH-particles can conveniently be used in the analysis of picomole amounts of lactate or pyruvate.