Partial purification and characterization by fluorimetry of lactoferrin of goats

PARTIAL PURIFICATION AND CHARACTERIZATION BY FLUORIMETRY OF LACTOFERRIN OF GOATS

Introduction: Lactoferrin (Lf) is a salmon red whey protein with a large molecular size of about 80 KDa, being found in milk and to a lesser extent in bile and tears. Lf is a cationic molecule that presents an isoelectric point (pI) around 8.0 to 8.5 having positive charges on its surface being this characteristic of paramount importance so that this protein can exert its biologicalactivities, and can be located in different mammalian species. Lf is known as a multifunctional protein, very important in the innate immunity system, because it can respond in various ways to physiological changes, present in several studies as a preventive and therapeutic treatment, because it presents antimicrobial, antifungal, immunomodulatory, antitumor, antiviral, neutralization of bioactive substances, among others. The workis to purify and characterize lactoferrin from goat's milk, monitoring purification by hydromyometry techniques. Materiais and methods: Skimmed milk was obtained by separating the fat from goat milk by centrifugation and acidified subsequently with HCl 0.1 M to pH 4,1, obtaining theacid serum. The acid serum foi neutralized with NaOH 0.1 M up to pH 6.8 and then centrifuged. A 50 mL rate of the sobrenatant submitted to Titration with NaOH 0.1N was removed up to pH8.0 and 8.3 and centrifuged afterwards. The utra aliquot of 50 mL was removed and submitted to saline precipitation profiles of 0-20%, 20-40%, 40-60% and 60-80% saturation of (NH₄) ₂SO₄. Fluorimetric analyses of isoelectric points and salt fractions were performed under excitation length conditions at 290 nm and emission wavelengths between 300-550 nm. Results and discussions: The isolation profile of Lf by (pI) pH 8.0, pH 8.3 presentor the fluorescence spectrum characteristic of lactoferrin (peak at 332 nm), while in the characterization by SDS PAGE 12%, using commercial lactoferrin (SIGMA) as astandard showing an isolation of the caprine Lf with the presence of other proteins. While the saline precipitation profiles of 0-20%, 20-40%, 40-60% and 60-80% saturation of (NH₄)₂SO₄ also presented the spectrum of fluorescence characteristic of lactoferrin. However, the profile of the precipitate resuspendidoof 40-60% showed the spectrum of fluorescence extinction characteristic of lactoferrin with higher protein concentration. Conclusion: In the present study, it was possible to partially purify caprine lactoferrin by means of isolation using precipitations by isoelectric and saline points with saturation with (NH₄) ₂SO₄ being monitored by hydromyometry.