Effects of gamma radiation on beta-lactoglobulin: oligomerization and aggregation.
The conformational changes and aggregation process of beta-lactoglobulin (beta-LG) subjected to gamma irradiation are presented. Beta-LG in solutions of different protein concentrations (3 and 10 mg/ml) and in solid state with different water activities (a(w)) (0.22; 0.53; 0.74) was irradiated using a Cobalt-60 radiation source at dose level of 1-50 kGy. Small-angle X-ray scattering (SAXS) was used to study the conformational changes of beta-LG due to the irradiation treatment. The irradiated protein was also examined by high performance size exclusion chromatography (HPSEC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing and reducing conditions and fluorescence. SAXS analysis showed that the structural conformation of irradiated beta-LG in solid state at different a(w) and dose level was essentially the same as the nonirradiated beta-LG. The scattering data also showed that the irradiation of beta-LG in solution promoted the formation of oligomers. Interestingly, from the data analysis and model building, it could be shown that the formed oligomers are linear molecules, built by linear combinations of beta-LG dimers (tetramers, hexamers, etc). The formation of oligomers was also evidenced by SDS-PAGE analysis and HPSEC chromatograms, in which products with higher molecular mass than that of the dimeric beta-LG were detected. Formation of intermolecular cross-linking between tyrosyl radicals are proposed to be at least partially responsible for this occurrence. From the results it could be shown that the samples irradiated in solution presented some conformational changes under gamma irradiation, resulting in well ordered oligomers and aggregates formed by cross-linking of beta-LG dimers subunits, while the samples irradiated in the solid state were not modified.
Oliveira
Cristiano L P
CL
Laboratório Nacional de Luz Síncrotron, Campinas, SP, Brazil.
de la Hoz
Lucia
L
Silva
Julio C
JC
Torriani
Iris L
IL
Netto
Flavia M
FM
D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D005720:Gamma Rays; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects*; D008958:Models, Molecular; D011489:Protein Denaturation / Q000528:radiation effects; D020836:Protein Structure, Quaternary / Q000528:radiation effects; D012996:Solutions; D013053:Spectrophotometry
D003432:Cross-Linking Reagents; D007782:Lactoglobulins; D012996:Solutions