Purificação parcial e caracterização por fluorimetria e espectrofotometria da Lactoferrina bubalina (Bubalus bubalis)

Abstract

Whey proteins have a high nutritional value, given by the presence of proteins with a high content of essential amino acids. Among the whey proteins, lactoferrin (Lf) is a glycoprotein with a molecular mass of approximately 80 kDa, present in mammalian whey, belonging to the transferrin family, it can be found in the milk of several mammals. In addition to having a specific ability to bind iron ion, Lf is a multifunctional protein having functions such as antibacterial, antiviral, antifungal, antiinflammatory and immunomodulatory activities. Thus, this work aimed to purify and characterize lactoferrin from buffalo milk, monitoring the purification by fluorimetry and UV-vis spectrophotometry techniques. 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 acidified whey. Then, the acid serum was neutralized with 0.1 M 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 ammonium sulfate (NH4)2SO4. Fluorimetric analyzes of the saline fractions were carried out under conditions of 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 (peak at 332 nm). The 40-60% resuspended precipitate was subjected to liquid chromatography on a Sephacryl S-100 gel filtration column. The spectrophotometric studies were performed with additions of 100 μL of 40-60% saline fraction (0.421 mg/mL), 100 μL of purified actoferrin (0.421 mg/mL) and 100 μL of amoxicillin (2.5x10-6 mol.L- 1). The UV-vis absorption spectra were recorded from 190 to 450 nm. Fractions 12 to 16 showed the characteristic fluorescence extinction spectrum of lactoferrin. 8% SDS-PAGE electrophoresis, using commercial lacteoferrin (SIGMA) as a standard, showed the presence of two protein bands in the standard range. Through the UV-vis absorption spectra studies, it was possible to detect that the interaction between lactoferrin and amoxicillin play an important role, decreasing the UV-vis absorption spectrum when compared to that of partially purified lactoferrin. Therefore, it was possible to isolate and partially purify lactoferrin buffalo by liquid chromatography in a gel-filtration column, the lactoferrin from buffaloes has a molecular weight greater than 87 kDa, it is possible to monitor the purification and characterization steps by fluorimetry and spectrometry.