Improved purification and fluorescence changes upon activation of human seminal plasma acidic protease proenzyme.

Modifications have been made to the previous purification procedure so that electrophoretically homogeneous acidic protease (EC 3.4.23.-) proenzyme of specific activity 800 units/mg may be isolated from human seminal plasma with a yield of over 50%. The intrinsic fluorescence of the proenzyme shows maximum excitation and emission wavelengths at 280 and 340 nm, respectively, typical of proteins containing tryptophan. Complete activation causes 30-35% decrease in intrinsic fluorescence, accompanied by a shift in gamma max to the blue of 4-6 nm. Time course studies indicate that acidification of proenzyme to pH 3.1 leads to a sudden large decrease in fluorescence that precedes both the appearance of active enzyme band on sodium dodecyl sulphate (SDS) polyacrylamide gels and the generation of enzyme activity as detected by the turbidimetric milk clotting assay. These results that acidification causes a rapid conformational change which promotes the release of the activation peptide from the proenzyme to yield the active enzyme.