Perfusion chromatography purification of a 15 kDa rice prolamin.
Prolamin extracted from rice flour using 55% n-propanol contained protein impurities. Reverse phase high-performance liquid chromatography (HPLC) on a perfusion column R2/H was used to separate rice prolamin from other proteins in less than 5 min. Prolamin eluted as the major peak. The isolated prolamin migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 4-12% Bis-Tris gel. Matrix-assisted laser desorption ionization mass spectrometry identified the rice prolamin as a 15 013 Da protein. The surface hydrophobicity (S(o)) of the HPLC-separated protein fractions was measured using the hydrophobic fluorescent probe PRODAN. A comparison was made with the surface hydrophobicity (S(o)) of corn prolamin and bovine serum albumin. Surface hydrophobicity values and solubility in 90% ethanol assisted in rice prolamin identification from other chromatographic peaks. The advantage of perfusion chromatography in purifying rice prolamin from other rice proteins included the reduced separation time, the speed at which the separation was carried, and the ability to regenerate the column in a short period of time and allow for more samples to be purified and separated.
Losso
Jack N
JN
Food Protein Biotechnology Laboratory, Department of Food Science, Louisiana State University Agricultural Center, 111 Food Science Building, Baton Rouge, Louisiana 70803-4200, USA. jlosso@lsu.edu
Bansode
Rishipal R
RR
Bawadi
Hiba A
HA
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005433:Flour / Q000032:analysis; D005456:Fluorescent Dyes; D008970:Molecular Weight; D012275:Oryza / Q000737:chemistry*; D010477:Perfusion; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D055315:Prolamins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D010940:Plant Proteins; D055315:Prolamins