Comparison between covalent and non-covalent BSA aggregation at acidic and neutral conditions

MARÍA DEL PILAR GUAUQUE TORRES; ANA E. LEDESMA ; CLAUDIO D. BORSARELLI

Congreso; XLIII Reunión Anual 2014; 2014

Covalent and non-covalent protein aggregation provides interesting immobilization strategies to obtain functionalized scaffolds for biocatalysis and biorecognition. In this report we compared the formation of non-covalent and covalent BSA aggregates formed in phosphate buffer solution at pH 3 and 7 by using several characterization techniques such as fluorescence, infrared spectroscopy, SEM microscopy and electrophoresis. Non-covalent BSA aggregation was achieved by heating at 60ºC, while covalent aggregation was done by formation of di-Tyrosine bridges with blue light photosensitization of a Ru(bpy) 3 2+ / S 2 O 82- mixture (1:20). Thermally-induced aggregation kinetics were monitored using ThT fluorescence, showed that nucleation time was reduced and the aggregation rate was increased at pH 3 compared to pH 7; indicating that BSA unfolded under acidic conditions favors the aggregation process. SEM images indicates that morphology of aggregates was controlled by pH, as at neutral pH spheroids of ∼ 100nm of diameter while at acid pH longer fibril-like shape aggregates (∼1µm), were formed. In the photosensitized covalent aggregation of BSA the initial rate of di-tyrosine formation was similar for acid and neutral pH, indicating that there is not influence of the conformational state of the protein. Nevertheless, after prolonged photolysis time there is also contribution to absorbance by changes in amino acid 1 O 2 -mediated. Both types of BSA scaffolds will be evaluated for enzyme immobilization.