Phosphorylation of the 12S globulin from rapeseed (Brassica napus L.) by phosphorous oxychloride: chemical and conformational aspects.
The effect of progressive phosphorylation by phosphorous oxychloride upon the conformation of the 300 kDa storage protein (cruciferin) from rapeseed has been studied using chemical analysis, SDS-PAGE, HPLC, analytical ultracentrifugation, viscometry, fluorescence spectroscopy, and hydrophobicity measurement. The amount of phosphorous in the protein increased with the excess of phosphorous oxychloride and the pH of reaction. The bulk of phosphorus was only loosely bound to the protein and was removed by washing with cold perchloric acid. The more stably bound phosphorus groups after reaction at pH 8 were found to be nearly equally attached to amino and hydroxyl groups, whereas phosphorylation at pH 10-11 led to predominant O-phosphorylation as detected by studying the acid- and alkali-lability of the protein-phosphorous bonds. A 50 kDa component appeared as a product of covalent cross-linking of the constituent alpha- and beta-polypeptide chains. A 2.5S fraction appeared as the main product of dissociation, which takes place after a critical step of modification. The higher the extent of phosphorylation, the larger was the percentage of higher molecular weight products, the percentage of which was most significant after modification under strongly alkaline conditions. They may be attributed both to products of chemical cross-linking and to noncovalently linked aggregates formed by interactions of partially unfolded derivatives exhibiting an increased surface hydrophobicity.
Schwenke
K D
KD
Institut für Angewandte Proteinchemie e. V., Stahnsdorfer Damm 81, D-14532 Kleinmachnow, Germany. food.proteins@prochem.pm.shuttle.de
Mothes
R
R
Dudek
S
S
Görnitz
E
E
D001937:Brassica / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005916:Globulins / Q000737:chemistry*; D006801:Humans; D010766:Phosphorylation; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014783:Viscosity
D005916:Globulins