Reversible conformational change in beta-lactoglobulin A modified with N-ethylmaleimide and resistance to molecular aggregation on heating.
beta-Lactoglobulin A (beta LG A) modified with N-ethylmaleimide (NEM-beta LG A) was purified by ion exchange chromatography, and modification of beta LG A by NEM was confirmed by time of flight mass spectrometry and 5,5'-dithiobis(2-nitrobenzoic acid) methods. The fluorescent spectrum of NEM-beta LG A was slightly different from that of native beta LG A. NEM-beta LG A gave no polymerization after heating at 80 degrees C and pH 7.5, as shown by polyacrylamide gel electrophoresis. Conformational change of NEM-beta LG A was observed at 80 degrees C by ultraviolet differential spectra, whereas after cooling it recovered to its original state as before heating, indicating apparent reversible thermal denaturation. Native beta LG A is resistant to pepsin hydrolysis, whereas heated beta LG A was easily hydrolyzed by pepsin. NEM-beta LG A before heating was also resistant to pepsin hydrolysis, and after heating NEM-beta LG A was still resistant to pepsin hydrolysis. These results indicate that NEM-beta LG A maintained a conformation similar to its native form even after heating. Addition of 0.2 M NaCl to the beta LG A heated under salt-free condition induced polymerization of heated beta LG A molecules, but not that of heated NEM-beta LG A. This seemed to indicate that the formation of inter- or intramolecular disulfide linkage made the heat-induced conformational change of beta LG A irreversible.
Kitabatake
N
N
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan. kitabata@food2.food.kyoto-u.ac.jp
Wada
R
R
Fujita
Y
Y
D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D008401:Gas Chromatography-Mass Spectrometry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007782:Lactoglobulins / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation
D004220:Disulfides; D007782:Lactoglobulins