Evaluation of assays for detecting alpha-1-protease inhibitor during purification from rat serum.
We purified the R1 alpha-1-protease inhibitor from rat serum and developed a convenient assay for its detection during purification procedures. Purification was accomplished by desalting, DEAE-Sephacel, zinc chelate, and reactive green-agarose columns. The resultant antiprotease had a molecular weight of 54,000 and inhibited elastase, chymotrypsin, and trypsin. By isoelectric focusing, five bands were produced with pI values from 4.3 to 4.7. Functional assays utilizing protease substrates imbedded in agarose plates were evaluated for the ability to distinguish the R1 alpha-1-protease inhibitor from the other serum antiproteases eluted in column chromatography fractions. This technique of screening for anti-protease activity was compared to conventional spectrophotometric methods and was found to correlate well when quantifying inhibition of elastase and chymotrypsin, but not trypsin. The presence of alpha-1-protease inhibitor was most reliably detected by testing for anti-elastase activity. Technician time and expense were saved by employing protease substrate plates to test chromatogrpahy fractions. This technique may facilitate purification of other protease inhibitors.
McGilligan
K
K
Department of Pediatrics, University of Southern California School of Medicine, Los Angeles.
Thomas
D W
DW
D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D005069:Evaluation Studies as Topic; D007526:Isoelectric Point; D008970:Molecular Weight; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin / Q000302:isolation & purification*
D001798:Blood Proteins; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; D002918:Chymotrypsin; D010196:Pancreatic Elastase