Active site-specific immunoassays
KG Mann, EB Williams, S Krishnaswamy, W Church, A Giles and RP Tracy
Department of Biochemistry, University of Vermont, Burlington 05405.
This study describes a process by which serine proteases that contain an
S-1 arginine subsite and active site histidine may be inactivated and
subsequently quantitated using a combination of peptidyl chloromethylketone
chemistry and immune recognition technology. Active site labeling and
inactivation of proteases is attained by modification of the active site
histidine with a peptidyl chloromethylketone. In the specific illustrations
demonstrated, we used the compound biotinyl-
epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This
reagent reacts quantitatively and specifically with the active site
histidine of a wide variety of proteases that are elaborated in the
coagulation and fibrinolytic system. The inactivated enzyme(s) may be
quantitated by combinations of antiprotein antibodies and avidin binding
technology using the biotin moiety on the peptide inhibitor. We have
demonstrated the capability of capture of inactivated enzyme products
directly on to solid-phase avidin with subsequent quantitation of bound
protein using specific antibodies. In the converse system we have captured
specific proteases using antiprotein antibodies in the solid phase and have
quantitated bound enzyme by using avidin. Subsequent detection and
quantitation has been achieved using the enzymatic activity of horseradish
peroxidase conjugated either to the antibody or to avidin. Both types of
assays are feasible, with avidin capture being the preferred mode when
enzyme is evaluated in the presence of excess zymogen, as would be common
in the evaluation of most blood-clotting enzymes. Assays are illustrated
for tissue plasminogen activator, plasmin, thrombin, factor Xa, and
activated protein C, which can measure protease concentrations as low as 50
pmol/L. Specific applications of the assays are provided in studies of the
activation of prothrombin by the prothrombinase complex and of factor X
with Russell's viper venom factor X activator. These assays measure the
mass of active site present in the reaction mixture and are relatively
independent of subspecies of enzyme or the environment in which the
activity is generated. These assay systems provide powerful tools for
elucidating product-precursor relationships in multienzyme feedback
reactions involving zymogen activation.
Volume 76,
Issue 4,
pp. 755-766,
08/15/1990
Copyright © 1990 by The American Society of Hematology
