I'm reporting on a novel technique that can be used to simultaneously measure activity levels for a panel of enzymes, such as the MMPs. The technique, termed the multiple-enzyme/multiple-reagent assay system (MEMRAS), relies on the use of reagents such as substrates with varying selectivity profiles against a group of enzymes. When reaction rates are measured by following a change in fluorescence with time, for mixtures of enzymes, an equation with unknown concentrations for each activity is generated for each reagent used. Simultaneously solving the set of equations leads to a solution for the unknown concentrations.
I have applied this mathematical technique to measure activity levels for mixtures of MMPs such as collagenase 3 and gelatinase A. In addition, because I was most interested in determining collagenase 3 levels as a potential biological marker for OA, I developed highly selective substrates for this enzyme by using results found in previous bacteriophage substrate-mapping experiments. Some of the best substrates tested have specific activities for collagenase 3 that are 37 000-, 17 000-, 90-, and 200-fold selective over stromelysin 1, collagenase 1, and gelatinases A and B, respectively.
Determination of kcat/Km for Dinitrophenyl (Dnp) Peptide Substrates. The Dnp substrates listed in Tables 1 and 2 were prepared and assayed as described below. Briefly, a dimethyl sulfoxide stock of 5 mM peptide was diluted in assay buffer so that the substrate concentration was 25 íM in a 200-íL volume. In the case of substrate 35, solubility proved to be a problem. Therefore, the substrate concentration was reduced to 5-10 íM. The assay buffer contained 50 mM Tris, pH 7.5; 200 mM NaCl; 5 mM CaCl2; 10 íM ZnCl2; and 0.01% Brij 35. After addition of enzyme (0.05, 0.17, 0.17, 10, and 17 nM final concentrations for collagenase 3, gelatinase A, gelatinase B, collagenase 1, and stromelysin 1, respectively), time points were taken at 10 min, 30 min, 1 h, and 2 h when a 50-íL aliquot was removed and quenched with 50 íL of 1% heptafluorobutyric acid (HFBA).
Reminds me of the duality nature of man. And stuff. Keep the faith brother!