An electrophoretic method for the detection of chymotrypsin and trypsin activity directly in whole blood

Abstract

In biomedical research and clinical diagnostics, it is a major challenge to measure disease-related degradative enzyme activity directly in whole blood. Present techniques for assaying degradative enzyme activity require sample preparation, which makes the assays time-consuming and costly. This study now describes a simple and rapid electrophoretic method that allows detection of degradative enzyme activity directly in whole blood using charge-changing fluorescent peptide substrates. Charge-changing substrates eliminate the need for sample preparation by producing positively charged cleavage fragments that can be readily separated from the oppositely charged fluorescent substrate and blood components by electrophoresis. Two peptide substrates have been developed for pancreatic alpha-chymotrypsin and trypsin. For the first substrate, a detection limit of 3 ng for both alpha-chymotrypsin and trypsin was achieved in whole rat blood using a 4% agarose gel. This substrate had minimal cross-reactivity with the trypsin-like proteases thrombin, plasmin, and kallikrein. For the second substrate (trypsin-specific), a detection limit of about 10-20 pg was achieved using thinner higher resolution 20 and 25% polyacrylamide gels. Thus, the new charge changing peptide substrates enable a simple electrophoretic assay format for the measurement of degradative enzyme activity, which is an important step toward the development of novel point-of-care diagnostics.

Whole blood assay for trypsin activity using polyanionic focusing gel electrophoresis

“…1A). Figures 1B and C show the basic steps of the trypsin assay that we previously developed in our first article on this technique [44]. The substrate is first mixed with a blood sample and allowed time to react with target enzyme in the blood and produce the positively charged cleavage fragment (Fig.…”

“…The trypsin substrate Ac-N-DGDAGRAGAGK-NH 2 (acetyl, Ac) was synthesized by Aapptec (Louisville, KY, USA) and subsequently labeled on the lysine residue's epsilon amine group with Bodipy FL-SE (Invitrogen, Carlsbad, CA, USA) following the manufacturer's standard labeling protocol (for details, see [44]). This formed the fluorescence-tagged substrate, Ac-N-DGDAGRAGAGK(epsilon amino (e)-Bodipy FL)-NH 2 .…”

“…As explained earlier in [44], this requires eliminating activity caused by endogenous enzyme, whose levels are unknown. This is accomplished by performing the cleavage reaction in buffer and then mixing with protease inhibitor-treated blood or plasma immediately prior to electrophoresis.…”

“…This is a consequence of the strong background absorption, significant autofluorescence and light scattering resulting from the various components within blood [43]. To address this issue, we recently demonstrated a simple, electrophoretic technique using charge-changing substrates that overcomes the limitation of previous assays and that facilitates measurement of protease activity directly in whole blood, without any sample preparation [44]. This assay uses negatively charged fluorescence-tagged peptide substrates that produce a positively charged fluorescent cleavage fragment (the signal) upon proteolysis.…”

A Sensitive fluorescent assay for trypsin activity in biological samples using BSA-Au nanoclusters

“…Among the reported methods for detecting enzyme activity, fluorometric analysis offers apparent advantage over other methods, such as electrophoretic method, 6,7 by virtue of their sensitivity and convenience. This method always relies on monitoring the fluorescence intensity as a result of enzymatic substrate proteolysis in the solution.…”