Precipitation of Hevea brasiliensis Latex Proteins with Trichloroacetic Acid and Phosphotungstic Acid in Preparation for the Lowry Protein Assay

Abstract

Many proteins derived from the latex of Hevea brasiliensis that remain soluble in trichloroacetic acid (TCA) can be precipitated by phosphotungstic acid (PTA). A combination of 5% TCA and 0.2% PTA precipitates a wide range of proteins effectively even when they are present in low concentrations (below 1 μg ml−1). In addition to its protein purification function, acid precipitation also increases the sensitivity of the subsequent protein assay by allowing the test sample to be concentrated. Another advantage of protein precipitation by TCA and PTA is that very small amounts of protein (of the order of 10 μg) can be repeatably recovered without the use of precipitate-bulking agents such as sodium deoxycholate. This general procedure of protein purification and concentration is simple and rapid, but the use of PTA may not be fully compatible with the Bradford protein assay. A modified Lowry microassay is described which enables about 3 μg ml−1 to be quantitated at the photometric absorbance of 0.05. When used in conjunction with protein concentration by precipitating with TCA/PTA, approximately 0.4 μg ml−1 protein present in 6 ml of solution can be assayed.