Banca de DEFESA: EDSON FERREIRA DA SILVA

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : EDSON FERREIRA DA SILVA
DATE: 02/09/2021
TIME: 14:00
LOCAL: PLATAFORMA GOOGLE MEET - VIDEOCONFERÊNCIA
TITLE:

PARTIAL PURIFICATION AND FLUORIMETRY CHARACTERIZATION OF LACTOFERRIN BUBALINE (Bubalus bubalis) AND EVALUATION OF THE INTERACTION WITH THE ANTIBIOTIC AMOXICILLIN AND THE FLAVONOID QUERCETIN.

KEY WORDS:

Lactoferrin, biological functions, purification, fluorimetry and spectrophotometry.

PAGES: 110
BIG AREA: Ciências Exatas e da Terra
AREA: Química
SUMMARY:

Lactoferrin (Lf) is a glycoprotein with a molecular mass of about 80 kDa. This enzyme belongs to the transferrin family that has a specific ability to bind iron. The molecular structure of Lf is made up of a single polypeptide chain and about 690 amino acid residues. Lf can be found in several mucous secretions, such as tears, saliva, gastrointestinal fluids, urine and seminal fluid, and in the secondary granules of neutrophils, being released in places where there is an inflammatory response. Lf 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 employ different purification techniques and compare the purification methods of lactoferrin-Lf present in buffalo milk, monitoring purification by fluorimetric techniques and evaluating the interaction of the protein with the antibiotic amoxicillin and the flavonoid quercetin (QCT) by UV-vis spectrophotometry. 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 was 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 purification 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%, 020-40%, 40-60% and 60-80% of 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 and fractions 12 to 16 showed the characteristic fluorescence extinction spectrum of lactoferrin. 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 of buffaloes and the commercial Sigma Lactoferrin fraction obtained a protein entity in the SDS-gel PAGE at 8%. Isoelectric purification by pI of lactoferrin buffalo from acidic 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. the precipitate resuspended at pH 8.3 obtained the characteristic fluorescence quenching spectrum of lactoferrin. The 9% SDS-PAGE electrophoresis showed the presence of entity bands in all fractions when compared to commercial lactoferrin. The spectrophotometric studies were performed with additions of 100 μL of partially purified lactoferrin buffalo fraction (0.842 mg/mL) and amoxicillin and QCT fractions at concentrations of 0, 1, 2, 3, 4, 5, 6, 7 and 8 µM. The UV-vis absorption spectra were recorded from 190 to 450 nm. The study showed the isolation and purification of lactoferrin buffalo by two distinct methods can be monitored by fluorimetry. Through the UV-vis absorption spectra studies it was possible to detect that the interaction between lactoferrin and amoxicillin play an important role, with an increase in the UV-vis absorption spectrum when compared to that of partially purified lactoferrin, quercetin (QCT) and amoxicillin.

BANKING MEMBERS:
Presidente - 426640 - FABIANE CAXICO DE ABREU GALDINO
Interno - 2089586 - FRANCIS SOARES GOMES
Interno - 1811274 - HUGO JUAREZ VIEIRA PEREIRA
Externa ao Programa - 1121304 - ADRIANA XIMENES DA SILVA
Externa ao Programa - 1121008 - SONIA SALGUEIRO MACHADO