Figure 2. Activity profile of VMA and iHA-VMA. A: Gelatin zymography showing gelatinolytic activity corresponding to Vibrio mimicus collagenase that remains active (VMA) and inhibitor of hyaluronic acid-VMA (iHA-VMA). B: Fluorescamine-based activity assay was performed by incubating 250 μg of gelatin in the absence (control) or presence of
VMA (1 µg), iHA-VMA (1 µg), or collagenase type IV (50 ng) in incubation buffer (pH 7.0) at 37 °C overnight, followed by a
change in pH to 6.0 using citrate buffer, and the addition of fluorescamine. Fluorescence was recorded; the relative fluorescence
intensity (RFI) is directly related to the gelatin degradation. Three independent experiments were performed, and data were
plotted with mean and standard deviation (SD). C: Collagen type 1 degradation (associated with decreased intensity of β, α1, and α2 chains of collagen) was assessed in the
absence or presence of 100 nU/μl VMA and iHA-VMA, respectively, on sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE; p<0.05, n.s., not significant).