Richard F. Parrish

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases

Abstract A number of amidino-substituted heterocycles were synthesized and examined for their inhibitory effect against arginine-directed esteroproteases, including human and bovine thrombin (EC 3.4.21.5), human factor Xa and plasmin (EC 3.4.21.7), bovine trypsin (EC 3.4.21.4), porcine pancreatic Kallikrein (EC 3.4.21.8), and boar acrosin (EC 3.4.21.10). Inhibition was competitive and reversible in all cases, and the K i values were taken to reflect binding conditions in the specificity pockets of the enzymes. A remarkably potent blocking agent was found in 5-amidinoindole. Compared to the established inhibitor benzamidine, it proved one to two orders of magnitude more effective against the majority of the proteases, the exceptions being trypsin and plasmin. Improved hydrophobic interaction, fortuitous hydrogen bonding, and charge transfer complex formation were considered in accounting for the considerable activity of the compound. Placement of the amidino moiety in the 6-position rather than in the 5-position on the indole ring resulted in a striking loss of inhibitory potency against human and bovine thrombin, factor Xa, and Kallikrein, yet slightly improved the affinity with respect to acrosin. Tightness of binding of the inhibitors to the enzymes was pH dependent and was significantly reduced below pH 7.0.

An apparent high molecular weight form of boar proacrosin resulting from the presence of a protein that binds to proacrosin

Abstract Gel chromatography at pH 3.0 demonstrated that a partially purified extract of ejaculated boar spermatozoa apparently contained two proacrosins with approximate molecular weights of 88.000 and 55.000. Disc gel electrophoretic experiments indicated that the high molecular weight form was actually a complex between the low molecular weight form and a protein with a molecular weight of 29,000. The fact that this complex did not dissociate at pH 3.0 indicates the need for caution in interpretation of data obtained by acidic chromatography of proacrosin preparations.

Identification of the in vivo sperm proacrosin into acrosin conversion sequence

Abstract 1. 1. Proacrosin and acrosin were quantitated in boar sperm which were incubated in ligated segments of gilt uteri and in buffer. 2. 2. Most of the proacrosin (70–80%) was converted into acrosin in sperm incubated in vivo for 120 min while less than 3% of the proacrosin was converted into acrosin in the control sperm. 3. 3. Electrophoretic comparison of the enzymatic activities in the sperm extracts to homogeneous m(inχ)-acrosin and m μ -acrosin suggested that the in vivo conversion sequence was: proacrosin → min(χ)-acrosin → m β acrosin.

Comparative synthetic substrate kinetics of porcine acrosin and porcine β-trypsin

Abstract 1. 1. Acrosin demonstrated a preferential binding of arginine containing substrates relative to lysine containing substrates and bound benzoyl containing substrates better than tosyl containing substrates. 2. 2. No clear cut pattern was observed in the maximal velocities of the acrosin catalyzed reactions. 3. 3. β-Trypsin manifested no selectivity for substrates containing arginine over lysine in either binding or maximal velocity, but greater maximal velocities were obtained for tosyl containing substrates relative to benzoyl containing substrates. 4. 4. The difference between the Kmapp, values for amide substrates and ester substrates indicated that acrosin hydrolysis proceeded through the classical double displacement mechanism.

Fluorescence and Immunodiagnostic Methods

In the past ten years, numerous applications of fluorescence methodsfor monitoring homogeneous and heterogeneous immunoassays have been reported. Advances in the design of fluorescent labels have prompted thedevelopment of various fluorescent immunoassay schemes such as the substrate-labeled fluorescent immunoassay and the fluorescence excitation transfer immunoassay. As sophisticated fluorescence instrumentation forlifetime measurement became available, the phase-resolved and time-resolved fluorescent immunoassays have also developed. With the current emphasis on satellite and physician’s office testing, future innovations in fluorescence immunoassay development will be expected to center on the implification of assay protocol and the development of solid-state miniaturized fluorescence readers for on-site testing.

INACTIVATION OF AMINATED HORSERADISH PEROXIDASE BY INTERACTION WITH S-SEPHAROSE

Horseradish peroxidase which had been aminated by periodate oxidation and reductive amination was purified by cation-exchange chromatography on S-Sepharose. Instead of the expected single peak of aminated enzyme, two distinct peaks of protein were eluted from the column. Evaluation of the protein in each of the two distributions showed that peak number 1 had spectral properties and specific activity similar to those of native enzyme. Distribution number 2 had a threefold reduction in the extinction in the Soret region at 404 nm and was completely devoid of enzymatic activity. This inactivation was caused by a specific interaction between the aminated peroxidase and the S-Sepharose matrix, resulting in a displacement of the heme prosthetic group out of its native orientation. The inactivation of the aminated peroxidase was found to be dependent on time, pH, and the support matrix itself. These results indicate that the S-Sepharose and Mono-S resins are not interchangeable, despite the chemical similarities of the two resins.