Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea.
Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.
Choi
Nack-Shick
NS
Proteome Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea.
Hahm
Jeung-Ho
JH
Maeng
Pil Jae
PJ
Kim
Seung-Ho
SH
D000818:Animals; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004586:Electrophoresis; D004795:Enzyme Stability; D005341:Fibrinolysin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents; D008623:Mercaptoethanol / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D011489:Protein Denaturation; D019163:Reducing Agents / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D003902:Detergents; D007202:Indicators and Reagents; D019163:Reducing Agents; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D014508:Urea; D017830:Octoxynol; D005341:Fibrinolysin; D004229:Dithiothreitol