Date of Graduation
Bachelor of Science
Chemistry & Biochemistry
Microdialysis is a diffusion-based sampling method that can be useful for monitoring various biological systems. Matrix metalloproteinases are a class of enzymes responsible for remodeling the extracellular matrix that, when dysregulated, are linked to various diseases. The delivery method of microdialysis is of particular interest as a sampling technique for enzymatic reactions. Microdialysis was performed in vitro using a model enzyme, porcine pancreatic elastase, because it is a useful substitute for matrix metalloproteinases. A colorimetric substrate for elastase, succinyl-ala-ala-ala-p-nitroanilide, and its product p-nitroaniline were measured using a UV-Vis spectrophotometer. Using an expanded Beer’s Law equation, both analytes’ concentrations were determined simultaneously from one dialysate sample using two of their overlapping absorbance wavelengths. The experiment aimed to test the effect flow rate, enzymatic solution concentration, and substrate concentration had upon the extraction efficiency of the procedure. Flow rate manipulations were consistent with literature, with higher flow rates yielding lower extraction efficiencies. Increasing the elastase concentration showed an increase in extraction efficiency of the substrate, whereas increasing the substrate concentration had no apparent effect on the extraction efficiency. Increasing either elastase or Succ-(Ala)3-p-NA concentrations, however, yielded higher p-NA concentrations.
Klucher, Justin M., "Enzyme kinetics studies to guide mathematical modeling of microdialysis sampling to predict in situ biochemistry" (2017). Chemistry & Biochemistry Undergraduate Honors Theses. 22.