Expanding the Use of Zymography by the Chemical Linkage of Small, Defined Substrates to the Gel Matrix

Abstract

In the postgenomic era, the comprehensive proteomic analysis of metabolic and signaling pathways is inevitably faced with the challenge of large-scale identification and characterization of polypeptides with a particular enzymatic activity. Previous work has shown that a wide variety of enzymatic activities of microbial, plant, and animal origin can be assigned to individual polypeptides using in-gel activity staining (zymography). However, a number of limitations, such as special substrate requirements, the lack of a standard procedure, and difficulties in distinguishing enzymes with overlapping activities have precluded the widespread use of zymography as a routine laboratory method. Here we demonstrate that, by employing small-defined substrates that are covalently attached to the gel matrix, we can largely overcome the aforementioned problems and assay readily a number of different classes of enzymatic activities within gels after standard SDS-polyacrylamide electrophoresis. Moreover, this development is compatible with the two-dimensional separation of proteins and thus has great potential in the high-throughput screening and characterization of complex biological and clinical samples.

Quantitative Enzyme Activity Determination with Zeptomole Sensitivity by Microfluidic Gradient-Gel Zymography

“…A consistent but less conservative value is offered by the x-intercept of Figure 4b at ∼4.0 zmol (0.57 fg, or on the order of 2500 CIP molecules). The approximate PLENZ LOD of 5 zmol (0.7 fg) compares favorably with both the low picogram sensitivities reported for macroscale zymographic techniques 48,49 and the value of 52 zmol reported by Wu et al for alkaline phosphatase in a capillary electrophoresis (CE) assay system. 56 However, a potential for optimization exists, as has been demonstrated by the application of laser-induced fluorescence detection in a CE system to study CIP kinetics down to the single molecule level.…”

“…Substrate Transport and Distribution. Postelectrophoresis enzyme assays in macroscale slab-gels are semiquantitative at best, 48,49 mainly due to the inability to quantify and control the substrate concentration at a given time and position in the gel. For example, when a small substrate molecule is incubated with the slab-gel, enzymemediated conversion of substrate to product is confounded by the kinetics of substrate diffusion from the gel surface to the true reaction site.…”