Soybean metal-binding proteins: calmodulin purification by hydrophobic interaction with polymer 3520.
Polymer 3520, a non-polar styrene divinylbenzene polymer, provides a simple way to purify calmodulin (CAM) from soybeans. This polymer, which selectively adsorbs CAM by hydrophobic interaction within the polymer matrix, contains no exchangeable groups; thus, interaction with CAM requires no Ca++ ions, and elution is achieved with 50% ethanol. Purification by this form of reversed-phase liquid chromatography is a substantial improvement over the conventional method, which requires high salt in elution buffers. CAM in soybean meal is first extracted with 80% ethanol in the presence of EGTA at room temperature and then chromatographed directly on a polymer 3520 column to yield pure CAM. Addition of non-ionic detergent (Nonidet P-40) to the ethanolic extract helps to separate extraneous proteins, lipids, sugars, and isoflavones. Such isolated CAM migrates as a single band during polyacrylamide gel electrophoresis and reversed-phase HPLC, and it retains activity stimulatory to phosphodiesterase.
Wang
L C
LC
Northern Regional Research Center, US Department of Agriculture, Peoria, IL 61604.
Peterson
R E
RE
Shang
K J
KJ
Ho
A K
AK
Rothfus
J A
JA
D000327:Adsorption; D002021:Buffers; D002147:Calmodulin / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000378:metabolism; D011137:Polystyrenes / Q000378:metabolism*; D011145:Polyvinyls / Q000378:metabolism*; D013025:Glycine max / Q000201:enzymology*
D002021:Buffers; D002147:Calmodulin; D011137:Polystyrenes; D011145:Polyvinyls; C051242:polymer 3520; D000431:Ethanol