Session: Undergraduate Research Posters: Biochemistry:

Use of crowding agents in enzyme kinetic studies enables the crowded in vivo cell environment to be mimicked in vitro. Since most intracellular macromolecular crowding is contributed by proteins, we have extensively employed bovine serum albumin (BSA) as an appropriate model crowder for our studies. Unfortunately, the strong UV absorption of BSA extends into the 340 nm region, resulting in unacceptable high background absorbance at this wavelength with elevated BSA concentrations. This limits the usable concentration range of BSA as a model crowder when measuring the kinetics of NAD(P)H-dependent enzymes at 340 nm. Development of a new assay for measuring NAD(P)H at longer wavelengths will enable use of higher BSA concentrations in macromolecular crowding studies. In the absence of BSA, water-soluble tetrazolium salt-8 (WST-8) is rapidly reduced by NADH to a formazan product absorbing at 450 nm with a pseudo first-order rate constant of 14.6 min^-1 at 25 ^oC, making this assay a viable candidate for our purposes. Introduction of BSA into this assay, however, yields a BSA concentration-dependent decrease in the reduction rate of WST-8 by NADH and a slow background rate of WST-8 reduction in the absence of NADH. These two issues must be resolved in order to implement this assay in a reliable enzymatic assay system. BSA contains 17 disulfide bonds and one free cysteine at position 34 (Cys34). Based on the ability of free thiols to reduce WST-8, we hypothesized that Cys34 may be a cause of at least some of the interference. Cys34 is also adjacent to one of the hydrophobic binding sites on BSA that may be a contributing factor in this interference. The impact of S-carboxymethylation of Cys34 as well as results of equilibrium binding studies to assess the potential role of BSA binding of assay reagent(s) will be presented.

David Seybert

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