A zone electrophoretic-isoelectric focusing study on the inactivation of human alpha 1-antitrypsin by elastase.
Previous experiments had shown that Alpha1-Antitrypsin (A1-AT) was rapidly inactivated by elastase in 30 min at 37 degrees C. Although the inhibitor moiety could be quantitatively accounted for, the elastase moiety was not recoverable. Side-by-side experiments under the above conditions (zero time and 30 min at 37 degrees C) using enzymatic and immunochemical (quantitative immunofixation-electro phoresis) methods, and four different purified A1-AT preparations, showed little or no increase in elastase concentration following degradation of the A1-AT elastase complex. Between 39 and 48% of the total elastase (in the complex) was undetectable either enzymatically or immunochemically. Elution-quantitation of the stained zones of sodium dodecyl sulfate polyacrylamide gel electropherograms of A1-AT elastase reaction mixtures where the inhibitor was present in excess of equivalence showed that the production of inactivated A1-AT occurs regardless of inhibitor concentration and that complex formation and inactivated inhibitor production are functions of elastase concentration. Isoelectric focusing (pH 4-6 gradient) of these reaction mixtures (zero time) showed the production of inactivated inhibitor under conditions of inhibitor excess and its transition from a nine zone pattern (2.3-fold molar excess of inhibitor; isoelectric points, 5.2 to 5.75) in the A1-AT elastase complex region to a six zone pattern (isoelectric points, 5.43 to 5.75) at equivalence. The six zone pattern was found to be a mirror image of the six zone pattern in the inactivated A1-AT region (isoelectric points, 4.53 to 4.91) with pH differences between zone pairs being practically identical.(ABSTRACT TRUNCATED AT 250 WORDS)
Baumstark
J S
JS
Department of Obstetrics and Gynecology, Creighton University, School of Medicine, Omaha, Nebraska 68131.
D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007120:Immunochemistry; D007525:Isoelectric Focusing; D010196:Pancreatic Elastase; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase