THE MOLECULAR WEIGHT OF ?-AMYLASE INHIBITOR FROM WHITE BEAN cv 858B (PHASEOLUS VULGARIS L.) IS 56 kDa, NOT 20 kDa

The molecular weights (Mrs) of α-amylase inhibitors (αAIs)fiom 18 (Ah) bean cultivars estimated by Superose 12 gelfiltration chromatography were 22–62% smaller than those determined by Sephadex G-100 gel filtration and by polyacrylamide gel electrophoresis (PAGE) methods. αAI-4 from WKB cultivar 858B was purified and the Mr was shown to be 51.0 kDa based on Sephadex G-100 gel filtration chromatography and by PAGE. A Mr for aAI-4 of 56.714 kDa was determined by laser-assisted time-of-flight mass spectrometry and appears to be the true Mr of the mature glycosylated active aAI-4. The results show that Superose 12 gelfiltration chromatography is not usefulfor Mr determination of some proteins. Sodium dodecyl sulfate electrophoresis (SDS-PAGE) showed that the 56.7 kDa aAI-4 molecule dissociated into 45.0, 33.6,15.2 and 12.4 kDa submolecules, with only the two small subunits, a and β, present at high SDS concentration. This provides evidence that the aAI-4 molecules composition is α2β2.