Buffalo (Bubalus bubalis) whey proteins conformational changes induced by vitamin B9 binding

LEANDRO FABIÁN BUSTOS; FRANCO EMANUEL VASILE; MARÍA ALICIA JUDIS; OSCAR EDGARDO PÉREZ

Conferencia; The 4th International Conference on Applied Biochemistry and Biotechnology (ABB 2021); 2021

Vitamin B9 is an essential nutrient for many human biochemical processes, such as cell multiplication, regulation of gene activity, production of red and white blood cells, etc. Therefore, its incorporation into foods and supplements, in the form of folic acid (FA), is a feasible alternative to achieve the recommended levels. However, this vitamin is easily degraded by the action of light, heat or oxygen. Among the strategies for its protection and delivery, the vehiculization of FA in protein-based nano systems, has been proposed. At this regard, it is important to characterize the ligand-carrier interactions and the derived structural modifications in the carrier. In this context, the objective of this work was to evaluate the conformational changes in the whey proteins of Bubalus bubalis induced by binding with folic acid. With this purpose, FA solutions were prepared at 0, 0.5, 1, 2, 5, 10 and 20 μM, in phosphate buffer pH 7 containing 5 μM of buffalo whey proteins, obtained from a protein concentrate (BWPC) with 55% proteins. For each system synchronous fluorescence spectra were obtained in the range 290 - 365 nm with Δλ of 15 and 60 nm. Additionally, IR spectra (4000 - 600 cm-1, 4 cm-1, 32 scans) were recorded in ATR mode. The baseline of the obtained spectra was corrected and the spectrum of the buffer + AF was subtracted. Next, the spectrum of the second derivate was obtained (Savitzky-Golay algorithm) and it was limited to the amide I band (1700 - 1600 cm-1). Finally, this spectrum was vertically inverted and the peaks were adjusted to Gaussian functions and associated with different components of the secondary structure of proteins. The synchronous fluorescence spectra showed a maximum emission (λmax) at 306 and 335 nm, for the microenvironments of the Tyr (Δλ= 15 nm) and Trp (Δλ= 60 nm), respectively. The addition of the vitamin caused the quenching of the fluorescence, 76.7% for Δλ= 15 nm and 52.8% for Δλ= 60 nm. Furthermore, the λmax of the Tyr shifted to higher values (red shift), indicating conformational changes and a decrease in the hydrophobicity. The λmax corresponding to the Trp microenvironment was not modified by the presence of the vitamin. The secondary structure of the protein fraction was composed by: 69.2% β-sheet, 6.7% β-turn, 2.3% α-helix and 13.6% random. The FA (20 µM) significantly increased (P