Effects of gamma radiation on β‐lactoglobulin: Oligomerization and aggregation

Abstract

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.

Structural modifications of β-lactoglobulin subjected to gamma radiation

“…There are no reported studies on either the conformational changes at secondary and tertiary structure level of irradiated b-LG, or how these changes influence the aggregation process. On the other hand, Oliveira et al (2007) verified that, when the protein was irradiated in the solid state (at different degrees of hydration, and at doses up to 50 kGy), the average size and compacity of the b-LG molecule did not change. These facts indicated that the conformational structure of the b-LG dimer was not affected by radiation (Oliveira et al, 2007).…”

“…The moisture content was 4.29 AE 0.02% and water activity 0.22 AE 0.01 (at 25 C). The preparation procedures of the b-LG samples with different water activities were described in Oliveira et al (2007).…”

“…In a recent study (Oliveira, de la Hoz, Silva, Torriani, & Netto, 2007), it was reported that, when irradiated with doses up to 50 kGy, native b-LG in solution (sodium phosphate buffer, 10 mM, pH 7.0) undergoes some structural alterations and, even though a compact structure is maintained, these alterations lead to an ordered aggregation, at least in part, via bityrosyl cross-links. There are no reported studies on either the conformational changes at secondary and tertiary structure level of irradiated b-LG, or how these changes influence the aggregation process.…”

Effects of low-dose ionizing radiation on α,β-globulins solutions studied by DSC

“…In practice doses, from 50 Gy up to kilograys are widely used, e.g., in radiotherapy treatment, in the manufacture of products derived from plasma (dose of 45 kGy inactivates all known blood-borne viruses), as well as in sterilization of the medical equipment and food products. The effects of irradiation at high doses of tens kGy on molecular, physicochemical, and functional properties of various proteins have been widely reviewed [2][3][4][5][6][7][8][9][10].…”

“…The influence of ionizing radiation on proteins has been the subject of great attention, and different techniques have been applied to study radiation-induced modifications in proteins [9,10]. Changes in peptide ions' abundance in serum after radiotherapy (51-72 Gy) as well as changes in serum proteome profiles are observed and expected to have a potential prognostic and predictive value in radiation therapy [18,19].…”