Chronopotentiometric Stripping Analysis of Gelatinase B, Collagen and Their Interaction

Abstract

Matrix metalloproteinases (MMP) belong to a group of zinc-dependent proteins that play a central role in the breakdown of extracellular matrices. Collagen, elastin, gelatin and casein are the main components of extracellular matrix cleaved by MMP. This paper aims to analyze the interaction between gelatinase B (MMP-9) and collagen using chronopotentiometric stripping analysis with adsorptive transfer stripping technique (AdTS CPSA). Under optimized experimental conditions (time accumulation of 90 s, supporting electrolyte 0.2 M acetate buffer pH 5, stripping current 1 mA), the detection limit (3 signal/noise) for MMP-9 was estimated as being 100 pM. The interaction between MMP-9 and collagen was studied according to the following scheme: i) HMDE surface was renewed. ii) Renewed surface of HMDE collagen (1 mg/mL) was accumulated for 90 s under open circuit. iii) The electrode was rinsed in ACS grade water and immersed in 5 mL drop of MMP-9. iv) The interaction between MMP-9 with collagen took place at open circuit. v) The electrode was then rinsed in ACS grade water. vi) The rinsed electrode was transferred into an electrochemical cell and measured in acetate buffer (pH 5). The CPSA signal of collagen after its interaction with MMP-9 increased more than 30% compared to that of only collagen. This increase in signal is likely due to the cleavage of collagen by MMP-9, hence its easy access to the electrodes surface.

Structural changes in metallothionein isoforms revealed by capillary electrophoresis and Brdicka reaction

“…5D. The decrease in the signal observed by DPV is in agreement with CE results because the precipitation of the molecules may disable the interaction of electrochemically active amino acids (mostly cysteine) with the electrode [50][51][52][53][54][55][56]. Using the same calculation process, the precipitation rate for MT-1 and MT-2 was determined as 125 and 37.9 ng of MT/min, respectively.…”

Chip gel electrophoresis as a tool for study of matrix metalloproteinase 9 interaction with metallothionein

“…For this purpose, we chose the method of differential pulse voltammetry Brdicka reaction (DPV Brdicka reaction) [21]. The following stock solutions were used for measurements: 1 mg bovine collagen (type-I) per mL (in the presence of 9% HCl [22]), MMP-9 10 mg/mL (ACS water), MT 10 mg/mL (ACS water). Primarily, Brdicka supporting electrolyte (1980 mL; 1 mM Co(NH 3 ) 6 Cl 3 and 1 M ammonia buffer (NH 3 (aq) 1 NH 4 Cl, pH 5 9.6)) was pipetted into the electrochemical vessel.…”

Nanotechnologies for society. New designs and applications of nanosensors and nanobiosensors in medicine and environmental analysis

“…This sensitive analysis can be carried out in the case of all peptides and proteins, which in their molecule contain cysteine. Among these molecules belongs also MTs [68,72,73,75,82,83,85], glutathione [174,[197][198][199][200][201], phytochelatins [199,200,[202][203][204] and others [205,206], which have been determined by using of stationary electrochemical instrument without autosampler.…”