NTU Undergraduates' research April 2014 - Biosciences | Page 74

Protein dynamics and solvent: Effect of
glycerol on domains motion

EDEN MANTAI
(N0361028)

Abstract
CPR is a membrane anchored flavoprotein of ̴78-kDa localized with its catalytic site at the
surface of the cytoplasm on the endoplasmic reticulum. CPR functions as an electron shuttle,
transferring electrons from NADPH through the FAD and FMN-coenzymes into a variety of
microsomal cytochrome proteins such as cytochrome c and cytochrome b5. The current
study investigate the effect of viscosity on the domain of cytochrome P450 reductase (CPR)
isolated from human fibroblast skin and 10 proline (10P) mutant CPR. The reduction of
cytochrome c by CPR was analyzed in a spectrophotometer using enzyme kinetic mode at
550nm for 2 min with measurement intervals at 5 seconds in the presence of 20%, 30%, 40%
and 50% glycerol at concentration different NADPH of 0.5 µM, 1 µM, 2 µM , 5 µM, 10 µM
20 µM, 40 µM, 80 µM and 160 µM.. The possibility that electron transfer rate through the
PCR is hindered by increased viscosity was measured. In agreement with this hypothesis, the
K m for cyto chrome c reductase was decreased in both wild-type and 10P mutant. The rate of
cytochrome c reduction was decreased as the concentration of glycerol increased, however
the data revealed an increased cytochrome c reduction with increasing NADPH
concentration suggesting the arise of reverse electron transfer.