Bovine Serum Albumin

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Introduction
Bovine serum albumin (BSA) is a transport protein in cow’s blood stream, similar to Human serum albumin in human blood. It binds different types of drugs for delivery to various parts of the body. One of these drugs is warfarin, a blood anticoagulant. This research project’s focus was on the interaction between BSA and warfarin derivatives; our assumption was that the protein binding site has different affinities for different warfarin derivatives; this was to be determined by comparing the equilibrium constants (Keq) and the Gibbs’ free energies of a number of warfarin derivatives. To study this, four warfarin derivatives were used, namely racemate mixtures of 2’-Fluoro warfarin (2’FW), 3’-Fluoro warfarin (3’FW), (R)-4’-Fluorowarfarin,
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This led us to hypothesize that fluorinating some positions on the phenyl ring might raise or lower the equilibrium constant of the reaction. A greater Keq would come as a result of the polar amino acids of the protein interacting with the new created polarity in the phenyl ring. On the other hand, the polar C-F bond might also disturb non-polar interactions between the non-polar amino acids and the non-polar C-H bond; this would result in a lower equilibrium constant.
It is clear from this experiment that the absorbance at 280 nm for BSA changes linearly between 100 and 200μM and becomes non-linear with increasing BSA concentration. This indicates severe concentration dependent changes in the optical properties of the protein. This could be due to either the refractive index changes in the sample or possible dimerization/aggregation. This change should be considered when performing the fluorescence analysis. This implies that the fluorescence titrations up to 200μM are well behaved i.e. linear. The experiment in figure 2 shows that when the samples are run in a 0.1 cm cuvette and not in a 1 cm one, and their corresponding absorbance multiplied by 10 to put them on the same scale, the curves do not simply align. This confirms that the changes at 200μM are a result of structural changes in the chromophore and not the analytical range

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