Matrix metalloproteinases (MMPs) play numerous roles in physiological and pathological processes including cancer. Interactions of MMPs with other zinc-binding proteins are of great interest mainly from the point of view of the participation of these proteins in carcinogenesis. The aim of this study was to utilize chip gel electrophoresis for investigation of matrix metalloproteinase 9 (gelatinase B, MMP-9) interactions with collagen. The interaction was observed and the effect of temperature was tested. We further focused on the study of activation of MMP-9 by the presence of zinc-binding proteins called metallothioneins (MT). We confirmed the basic presumption of the activation of MMP-9 by the presence of MT similar to the temperature effect. Moreover, we chose the method of differential pulse voltammetry Brdicka reaction to verify our results. It can be concluded that the activity of MMP-9 was higher in the presence of MT in comparison with the experimental collagen(type I)-MMP-9 mixture in vitro.