Purificação parcial e caracterização por fluorimetria de lactoferrina de bubalinos (Bubalus bubalis) e avaliação de sua interação com fármacos

Abstract

Lactoferrin (Lf) is a glycoprotein with a molecular mass of about 80 kDa, belonging to the transferrin family, having the specific ability to carry out binding with the iron ion. Present in mammalian milk, it is considered a multifunctional protein, playing several biological roles, such as antibacterial, antiviral, antifungal, anti-inflammatory, antitumor, antioxidant and immunomodulatory activities. This work aimed to purify and characterize lactoferrin from buffaloes and evaluate the protein-antibiotic and protein flavonoid interactions. The processing of buffalo milk began with the separation of fat by centrifugation. The skimmed milk was acidified with 0.1 M HCl to pH 4.6 obtaining the acidified whey. At this stage, the processing of acid whey is subdivided into two distinct processes: the processing of the acid whey for purification by chromatography and the processing of the acid whey for isolation by the isoelectric point of lactoferrin. In the purification protocol by liquid chromatography, the acid serum was neutralized with NaOH to pH 6.8 and the supernatant obtained by centrifugation was subjected to saline precipitation in the profiles of 0-20%, 20-40%, 40-60% and 60-80 % saturation of (NH4)2SO4. Fluorimetric analyzes of the saline fractions were performed at excitation length at 290 nm and emission wavelengths between 300-550 nm. The saline profile of the 40-60% resuspended precipitate showed the characteristic fluorescence extinction spectrum of lactoferrin. The 40-60% resuspended precipitate was applied to liquid chromatography on Sephacryl S-100 gel filtration (size (1x60 cm) with a flow of 0.1 mL/min, bed volume of 50 mL in 0.2 M phosphate buffer pH 7.7 ). Fractions 12 to 16 showed the characteristic fluorescence extinction spectrum of lactoferrin. Isoelectric purification of the pI of lactoferrin acid serum was titrated to pH 5.2 with 1 M NaOH, the supernatant obtained by centrifugation was titrated to pH 8.3 and centrifuged. The samples of the resuspended supernatants and precipitates were subjected to the quantification of protein content by the Bradford method and the fluorimetric and electrophoretic studies were carried out. UV-vis absorption spectroscopy studies of lactoferrin with amoxicillin and quercetin were performed. The 8% SDS-PAGE electrophoresis obtained a better charge/mass resolution of the dye labeled bands, which shows the presence of two protein entities in the partially purified lactoferrin fraction and a band in the commercial bovine lactoferrin standard in the 8 SDS-PAGE gel %. The precipitate resuspended at pH 8.3 from the isoelectric purification obtained a higher 75 KDa protein band consistent with the commercial bovine lactoferrin standard. The study showed the purification of lactoferrin buffalo by two different methods and the monitoring by fluorimetry of the purification process of lactoferrin buffalo, as well as the UV-vis absorption studies of lactoferrin with drugs showed that the complexation of these substances can generate changes in the structure of the protein the results suggest that the addition of drugs changes the polarity around the protein in that some residues such as Trp and Tyr are exposed to a more polar environment, while some external residues are embedded in globular proteins.