Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting.
Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the alpha subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).
Sweadner
K J
KJ
Neurosurgical Research, Massachusetts General Hospital, Boston 02114.
D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D004355:Drug Stability; D004586:Electrophoresis / Q000379:methods; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry; D007700:Kinetics; D008861:Microsomes / Q000737:chemistry; D010196:Pancreatic Elastase / Q000378:metabolism; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis; D051381:Rats; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D014357:Trypsin / Q000378:metabolism*; D014360:Trypsin Inhibitor, Kunitz Soybean / Q000378:metabolism*
D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D014360:Trypsin Inhibitor, Kunitz Soybean; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin