This paper describes an undergraduate biochemistry laboratory module consisting of a set of experiments designed around a purification scheme for bovine serum albumin (BSA). Students purify BSA from cow plasma by a combination of salt and acetone precipitation, equilibrium dialysis, ion exchange, and size exclusion chromatography. Students use the bromocresol green–BSA complex assay to quantify albumin at each step in the scheme and generate a purification table. In addition, trace amounts of IgG’s and nucleases, the major contaminants in BSA purification, are probed by Western blotting and DNase assay, respectively. This module exposes students to the principles and techniques of separation of proteins based on solubility, size, and surface charge. In addition, students learn the concept of limit of detection of analytical techniques such as SDS-PAGE and Western blotting. The module utilizes two 3-h laboratory periods, with a 1-h prelab lecture per week, for about 6 weeks. Depending on class size, groups consist of two to four students. The module is suitable for students who have completed a semester of biochemistry. Instructors can extend the module to advanced level laboratory by including enzyme kinetics and protein structure–function studies based on albumin’s pseudoesterase activity and intrinsic tryptophan fluorescence, respectively.
Benzofuran‐2‐yl methyl ketone (BMK) is a prochiral ketone that is reduced to benzofuran‐2‐yl ethanol (BMA) when exposed to tissues of certain plants. The process of using biological tissue to convert one compound to another, typically with high enantiomeric excess, is called a biotransformation reaction. Because different plants catalyze this conversion, the primary goals of this research were to identify plants that are successful in conversion, determine an approximate time necessary for each plant to perform the conversion, and then analyze the product of each plant to determine the enantiomeric excess. Optical purity of BMA was determined using polarimetry. The BMA produced by carrots had a [α] = −16.6 which agrees with the literature value for the S‐isomer of BMA. However, the BMA produced by potatoes had a [α] = −.74 which may indicate a mixture that is nearly racemic. To explore the reaction further, antimicrobial studies were carried out adding either BMK or BMA to YPD agar plates growing with Baker's yeast. In these preliminary studies, S‐BMA, produced by carrots, consistently demonstrated a greater growth inhibition of the Baker's yeast than BMK. The growth inhibition of the yeast by BMA was dose dependent. The antimicrobial studies were also expanded to BL21 E. coli grown on LB agar, showing similar results to those of yeast. The antimicrobial activity of S‐BMA against yeast and bacteria was also compared to that of the racemic mixture produced by potato, which showed no significant differences between the two. The antifungal activity of BMA against yeast was then compared to the common over‐the‐counter antifungal compounds miconazole, clotrimazole, terbinafine and tolnaftate. The latter two had no effect on growth, but miconazole and clotrimazole inhibited yeast growth substantially. The antibacterial activity of BMA against E. coli was compared to ampicillin. In these preliminary antimicrobial studies, BMA has shown comparable activity to common compounds.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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