Papain, a sulfhydryl protease has been immobilized on flat-sheet modified polysulfone membranes and hydroxyethyl cellulose coated polyethersulfone hollow fibers. Amidase activity of the enzyme in solution and on the membranes has been assayed. Immobilized papain on the modified polysulfone membrane and the hollow fibers retains 12% and 25% of its activity (with 1 mmol dm(-3) substrate) in solution, respectively. Loading experiments revealed decreased activity on the modified polysulfone membrane with increased enzyme loading. Adsorption experiments for the reaction product, p-nitroaniline, have been performed and an attempt has been made to correct for this in activity calculations. Apparent Michaelis-Menten parameters were determined for the modified polysulfone and hollow fibers, with both K-m and V-max being lower in the immobilized cases. Electron paramagnetic resonance study of the changes in active site conformation of an enzyme on a hollow fiber membrane are reported for the first time. Experiments using the sulfhydryl-specific (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl)methanethiolsulfonate spin label depicted the presence of two subpopulations of immobilized papain on the hollow fibers, one of them active and one denatured.