Molecular Weight of Proteins Determined by Superose 12 Column Chromatography May Not be Correct

摘要

Our research on α-amylase inhibitors (αAIs) and other proteins indicated that true molecular weights cannot be determined by Superose 12 column chromatography. In this paper, we use data on the molecular weights of red kidney bean (RKB) and white kidney bean (WKB) α-amylase inhibitors (RKB αAI and WKB αAI) and five standard proteins (cyto-chrome c, Kunitz soybean trypsin inhibitor, chicken ovalbumin, bovine serum albumin, and human transferrin) to document this problem. Homogeneous RKB αAI from red kidney bean (Phaseolus vulgaris) seeds was used to determine molecular weight (MW) using three methods. The molecular weight of purified RKB αAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis and Superose 12 gel filtration methods were 49.0, 51.0 and 22.9 kD, respectively. Purified RKB αAI, along with five standard proteins (above), were used also to test the effect of ionic strength in the running buffer on molecular weight determination using the Superose 12 column. The elution volume and Vn/Vo for human transferrin, bovine serum albumin, chicken ovalbumin and Kunitz soybean trypsin inhibitor increased to a plateau when the ionic strength in the running buffer increased. The Vn/Vo for cytochrome c and molecular weight for RKB αAI initially increased from 0 to 0. 05 M NaCl but then decreased at higher NaCl concentrations (the apparent size of RKB αAI was affected more than was that of cytochrome c). The determined MW for RKB αAI and cytochrome c were different when different ionic strength buffers were used. In all cases for RKB αAI the MWs were 22-62% smaller than those determined by Sephadex G-100 gel filtration and by electrophoresis. The data indicate there is ionic interaction between the proteins and Sephadex 12 in low i-onic strength buffers and hydrophobic interaction at higher ionic strength buffers. The results gave bell-shaped plots; however, even at the top of the bell the molecular weights were also too low. Researchers should be cautious when using a Superose 12 column for molecular weight determinations.