Real-time dual zymographic analysis of matrix metalloproteinases using fluorescein-isothiocyante-labeled gelatin and Texas-red-labeled casein

Abstract

Zymography is a widely used technique for identifying the proteolytic activity of enzymes [1–3]. We recently developed a real-time zymographic system for matrix metalloproteinases (MMPs) using fluorescein-isothiocyanate (FITC)-labeled gelatin as a substrate [4]. In this report, we describe an improvement of this technique wherein both Texas-red-labeled casein and FITC-labeled gelatin are incorporated into a single polyacrylamide gel as substrates. The digestion of gelatin and casein by different MMPs is independently observed on the gel using a transilluminator with the appropriate optical filters. With this fluorescent detection system, MMP-3 could be distinguished from MMP-2 and MMP-9 on the same gel and activities could be measured concurrently and in real time. Using this dual detection system, we can save the time and reagent for the experiment and the precise comparison of the digestion bands is accomplished as well. Methods. Collagen was extracted from newborn calf skin by pepsin digestion and purified by sequential salt precipitation [5]. Collagen was labeled with FITC (Dojindo Laboratories, Kumamoto, Japan) in a 0.5M sodium carbonate–sodium hydrogen carbonate buffer (pH 9.5) as previously described [4]. The absorbance at 495 nm for the purified 0.2% FITC–collagen in 1% sodium carbonate solution was 0.939. Calculation from the molecular extinction coefficient of FITC (77,000 at 495 nm) indicated that 1.83 molecules of the FITC bound to each molecule of type I collagen (300 kDa). FITC–collagen was denatured into FITC–gelatin by heating in boiling water when it was used as a substrate for zymography. Commercial casein was from New Zealand Milk Products (Reporoa, New Zealand). A total of 200ml of 0.25% casein was prepared in 0.5M sodium carbonate– sodium hydrogen carbonate buffer, pH 9.0. Ten milligrams of Texas red (Research Organics, OH) was directly added to the casein solution with gentle stirring [6]. The conjugation reaction was carried out at 4 C for 5 h in the dark. We added 6N HCl to the mixture until the pH reached 4.6 (the isoelectric point of casein) to stop the reaction and to precipitate the labeled casein. The precipitate was then dissolved in a solution of NaOH (pH 9.4). The Texas-red-labeled casein was purified by repeating this precipitation–dissolution cycle five times. Next, the purified Texas red–casein was lyophilized. The absorbance at 596 nm for Texas red–casein (0.05%) in the NaOH solution (pH 9.0) was 0.201. Based on the molecular extinction coefficient (84,000 at 596 nm), 0.10 molecules of Texas red were bound to each molecule of casein (27.8 kDa). The sodium dodecyl sulfate–polyacrylamide gel electrophoresis system used was based on the method of Weber and Osborn [7] with several modifications [4]. Zymography was performed under nonreducing conditions in an 8 7 cm (0.75-mm thickness) slab gel containing 12% acrylamide, 0.25mg/ml FITC-labeled gelatin, and 0.5mg/ml Texas-red-labeled casein. A standard stacking gel (5% acrylamide) was used. MMPs in sample buffer (50mM Tris–HCl, pH 6.8, 1% SDS, 0.1% bromphenol blue, and 30% glycerol) were loaded onto a gel without heat-denaturation.The samples were run at a constant voltage (110V) for approximately 2 h with cooling in a water bath (at approximately 10 C). After electrophoresis, the gel was washed four times Analytical Biochemistry 307 (2002) 390–392