Mary McPhail

Date Approved

2005

Degree Type

Open Access Senior Honors Thesis

Department or School

Chemistry

First Advisor

Dr. Steven Pernecky

Second Advisor

Dr. Heather Holmes

Abstract

The purpose of this project was to develop an upper level undergraduate biochemistry laboratory (CHEM 453) based upon a novel research project, designed to replace an existing instructional laboratory that employs ‘cookbook’ exercises. Methods for the expression, purification, spectral analysis of structural integrity, and electron transfer capability of rabbit cyt b5 were adapted from published work done by Dr. Lucy Waskell of the University of Michigan and Dr. Mansuy of the University of Paris. In our hands, rabbit liver cyt b5 expressed in E. coli was purified to 45% specific content of the theoretical content. Additionally, 37 nmol cyt b5/mg of protein was found using a Biuret Standard Assay giving a 63% specific content from the theoretical value of 58.8 nmol cyt b5/ mg protein. The specific content is the amount of cyt b5 (holo and apo) that is present out of all the protein present. Therefore, the difference of the 18% could be due to cyt b5 or another protein. In the CHEM 453 lab purification of the cyt b5 gave a specific content that was 70% of the theoretical value, the same as was obtained previously by Waskell. The rate constant for tetrahydrobiopterin reduction of cyt b5 was determined to be 0.005 M-1cm-1 compared to Mansuy’s kinetic rate constant of 0.20 M-1cm-1. This discrepancy might be due to differences in concentration of cyt b5 , the reductant, or to the fact that Mansuy used a trypsonized form of cyt b5 whereas we used the intact rabbit cyt b5 . Reduction was confirmed to be a second order process. The research effort was successful in preparing a research-based experiment for the CHEM 453 course.

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