Methods: β-Lg (donated by Davisco Inc.), was hydrolyzed with bromelain (3% protein w/w in distilled water, 25 U enzyme g-1 of substrate, pH 7.5, 240 min) and then polymerized by TG (7% hydrolysate, 10 or 25 U TG g-1 protein, 50 ºC/180 min). When polymerization was carried out pre hydrolysis, β-Lg (7% w/w) was polymerized by TG (10 U TG g-1 protein, 50 ºC/180 min) in 0.1 M Cys or after heat treatment (80 ºC/60 min), and then hydrolyzed under the same conditions previously described. The hydrolysis reaction was monitored by pH-stat method and the samples were evaluated by SDS-PAGE/tricine. Antigenicity was evaluated by Immunoblotting and ELISA assays using sera from mice sensitized with β-Lg (IgE anti-β-Lg).
Results: The treatment with TG (10 or 25 U TG g-1), post hydrolysis, lead to formation of products with a wide molecular weight (MW) distribution (3 to 26 kDa), and other products with high MW, indicating partial polymerization. The samples obtained from polymerization pre hydrolysis showed bands with low MM (< 6.5 kDa) and also products > 26 kDa, indicating a partial hydrolysis of the polymers. Immunoblotting analysis showed no reaction towards specific IgE with any of the samples. ELISA assay showed that the IgE-binding response to sample polymerized by TG post or pre hydrolysis with bromelain were significantly reduced (IgE ≤ 35.21 ug mL-1) as compared to untreated β-Lg (IgE 216.20 ug mL-1 ± 27.58) (P<0.05).
Conclusions: : These results suggested that hydrolysis with bromelain combined with polymerization by TG was capable of reducing the antigenicity of β-Lg, being a potential method for modifying the antigenic properties of proteins.
Acknowledgements: CNPq and FAPESP for financial support.