Abstract

The TBA (thiobarbituric acid) Assay is commonly used for quantifying the products of biochemical processes, such as degradation of pectin by pectate lyase, and lipid peroxidation by free radicals. Pectate lyase cleaves the polysaccharide pectin (found in the cell walls of plants), effecting the degradation of plant tissue. Lipid peroxidation occurs via a radical chain reaction initiated in the cell by reactive oxygen species, which can be harmful. The TBA assay relies on absorbance spectrometry measurements of the colored products of these two processes. While studying the activity of pectate lyase, we found that the absorbance of the final TBA adduct varies widely with the temperature of the sample. Therefore, modulation of sample temperature is important to collecting accurate data, and failure to do so will generate experimental error for labs using this procedure. On the opposite side of the enzyme activity coin, we also explored conditions for, and methods of, quantifying protein denaturation. We used the aggregation of insulin as a proxy for measuring its denaturation by various insults, and found that two types of aggregation can occur: “flaking” or “fogging.” We found that fogging is the more useful type of aggregation for measurement by absorbance spectrometry. We therefore optimized the conditions for generation of a fog. This insight may allow for future studies of the stability of insulin and other proteins.