Effects of substrate and separation method on acrosin amidase measurements

The purpose of this study was to increase the accuracy and reproducibility of the acrosin amidase assay and to assess the effects of different methods of sperm isolation on total sperm acrosin activity. Specific acrosin activity was measured by the procedure described by Kennedy et al (1989) comparing the usual substrate, N-alpha-benzoyl-DL-arginine-p-nitroanalide hydrochloride (DL-BAPNA) with the L-(L-BAPNA) and D-(D-BAPNA) isomers. Activity measurements were also compared on sperm isolated by methods: (1) centrifugation through buffered Ficoll, (2) method 1 plus an additional wash in buffered Ficoll, (3) back addition of supernatant from method 1 to spermatozoa isolated by method 2, and (4) swim-up into synthetic human tubal fluid media (mHTF) and using L-BAPNA. The specific activity of acrosin was dependent on substrate concentration up to 2.1 mM DL-BAPNA and 2 mM L-BAPNA. The maximum reliable solubility of DL-BAPNA was approximately 2.1 mM in 10% dimethylsulfoxide (DMSO):90% detergent buffer. There were no solubility constraints for L-BAPNA through 6.3 mM (> 5 times Km). D-BAPNA (1 mM) was not hydrolyzed by acrosin. Mean specific acrosin activity was higher using 6.3 mM L-BAPNA (159 +/- 11.4 microIU/10(6) sperm) than with 2.1 mM DL-BAPNA (81.4 +/- 10.9 microIU/10(6) sperm; P < 0.001, n = 16). Sperm isolated by methods 2 and 4 had higher specific acrosin activity than sperm isolated by method 1 (P = 0.002). Sperm treated per method 3 had similar acrosin activity as sperm isolated by method 1 (140 +/- 14.1 vs. 149 +/- 13.8 microIU/10(6) sperm). The K(m) for acrosin, calculated through 6.3 mM L-BAPNA, was 0.6 microIU/10(6) sperm. L-BAPNA is superior to DL-BAPNA as substrate for a clinical acrosin assay, increasing the reproducibility and accuracy of the assay. Simple Ficoll separation is not completely effective at removing acrosin inhibitors and additional separation steps may be necessary to assess true acrosin activity.