Date of Graduation

5-2015

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor

Stenken, Julie A

Reader

Kumar, Suresh

Second Reader

Kilyanek, Stefan M

Third Reader

Dempsey, Sean A

Abstract

Microdialysis is a diffusion-based method used to collect analytes from specific locations in the body. Matrix Metalloproteinases (MMPs) are important enzymes located throughout the body that play a role in the breakdown of extracellular matrix. Microdialysis sampling in conjunction with appropriate analysis methods can be used to observe the reactions of MMPs in real time. This project is aimed at determining the activity of porcine pancreatic enzyme, a known substitute for MMP-12, using nanodrop spectroscopy. This is a necessary pre-requisite toward using microdialysis in an in vivo imaging setup. These experiments were performed using Succinyl(Ala)_3-p-nitroanilide (suc(Ala)3-p-NA), 4-nitroaniline, and porcine pancreatic elastase. UV-Vis spectroscopy was a suitable measuring device, since suc(Ala)3-p-NA and 4-nitroaniline have maximum absorbance separated by 65 nm. The 100kDa molecular weight cutoff (MWCO) probe allowed for the enzyme to diffuse into the collection vials, causing elevated recovery of the p-nitroaniline. This meant that a 20kDa MWCO membrane had to be used. As with most microdialysis, at slower flow rates, the collection of p-nitroaniline increased, collecting 49.5 μM at 1μL/min and 18.7 μM at 5μL/min using the 20kDa MWCO probe. Using UV-Vis it was possible to eliminate the overlapping absorbance values of the suc(Ala)3-p-NA and p-nitroaniline.

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