Studies of complexes between proteases, substrates and the protease inhibitor alpha 2-macroglobulin using capillary electrophoresis with laser-induced fluorescence detection.
Capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection is shown to constitute a unique technique for the investigation of the interaction between proteases, protease inhibitors and substrates. Under optimized analysis conditions, the formation of a complex between FITC-labelled proteases such as trypsin, plasmin, alpha-chymotrypsin and the (unlabelled) protease inhibitor alpha 2-macroglobulin was studied. This is not possible with UV detection, since under such conditions the complex cannot be distinguished from the unreacted protease inhibitor. Low ratios of FITC bonded to the proteases further complex formation, while high ratios often prevent the reaction. Complex formation shows a strong dependence on the incubation conditions (pH, salt concentration, temperature, incubation time). Once formed, however, the complexes are stable under CZE conditions (e.g., a pH of the electrophoresis buffer of 10.5) for at least 30 min. Treatment with sodium dodecyl sulfate (5 min at 90 degrees C or 30 min at 75 degrees C) does not destroy the complexes, whereas treatment with mercaptoethanol (reduction of disulfide bonds) eliminates the peak from the electropherogram. Both findings argue for the formation of a covalent bond between the protease and the inhibitor during complex formation. Since the reaction of the proteases with alpha 2-macroglobulin does not involve the binding site of the former, a residual proteolytic activity is still observed in the ensuing complex. The extent of the inhibition of the remaining trypsin activity in a trypsin--alpha 2-macroglobulin complex was established to depend on the molecular mass of the second trypsin inhibitor.
Reif
O W
OW
Institut für Technische Chemie, Universität Hannover, Germany.
Freitag
R
R
D019075:Electrophoresis, Capillary; D010450:Endopeptidases / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate; D007834:Lasers; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000737:chemistry*
D014361:Trypsin Inhibitors; D000511:alpha-Macroglobulins; D010450:Endopeptidases; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate