Antiretroviral activity of Pterois volitans (red lionfish) venom in the early development of human immunodeficiency virus/acquired immunodeficiency syndrome antiretroviral alternative source

Aim: This study aimed to investigate the antiviral activity of Pterois volitans phospholipase A2 (PV-PLA2) from Indonesia to human immunodeficiency virus (HIV). Materials and Methods: Fresh venomous fin parts of wild PV specimens were collected from Java Sea waters. Then, it washed using phosphate buffer pH 7.0 and immersed in phosphate buffer pH 7.0 0.01 m containing CaCl2 0.001 m for 24 h. The immersed fin then allowed for extraction process by sonicating for 2×8 min with 80% pulse and 20 kHz output with temperature controlling to avoid denaturation. The crude venom (CV) extracted from the fin is allowed for purification by 80% ethanol (ET) precipitation and ammonium sulfate fractionation method. The purified PV-PLA2 then analyzed using Lowry's method, Marinette's method, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide assay. After determining the purest and safest sample of six samples analyzed, the chosen sample then tested into simian retrovirus-2 (SRV2)-A549 culture (48×104 cells/mL at 1-4 ppm), and compared to the CV sample (1-4 ppm) and lamivudine (100 ppm). The culture then is analyzed using a quantitative real time-polymerase chain reaction to find out the copy number of SRV-2 virus in each culture. Results: The protein's activity, concentration, and purity analysis revealed that the PV-PLA2 purified using ammonium sulfate fractionation has the highest activity (1.81 times higher than the CV at 80% fractionation) and has higher purity than the sample from ET fractionation. The testing of the sample purified using ammonium sulfate fractionation at 80% saturation level shown that it has a 97.78% inhibition level toward SRV2-A549 culture at 4 ppm. However, in comparison to lamivudine which has 99.55% inhibition level at 100 ppm, it needs much lower concentration to achieve the same result. Conclusion: The significant inhibition of SRV2-A549 culture shown that the PV-PLA2 extracted from PV venom has the potential to become anti-HIV substances. It would be worthwhile to further evaluate the antiretroviral activity of PV-PLA2 in the in vivo studies.

Purification of a Fibrinolytic Protein from the Yellow Mealworm Beetles, Tenebrio Molitor

A novel of fibrinolytic protein was isolated from the Tenebrio molitor, using three purification steps (ammonium sulfate fractionation, DEAE-32 cellulose chromatography column, and SephadexG-75 dextran gel column). After testing, the protein from Tenebrio molitor has fibrinolytic activity, and it relative molecular mass was 56.1 kD.

Preliminary Fractionation and Derivatives Changes of Myoglobin from Tilapia (O. niloticus♀×O. aureus♂) Dark Muscle with Ammonium Sulfate

Ammonium sulfate fractionation was implicated as one of essential steps to purified Myoglobin (Mb).We adopted SDS-PAGE analysis and absorption spectra scanning to demonstrate the effect of ammonium sulfate on Mb and its derivatives. The results shown that protein with the molecular weight above 37.8kDa were dominated in myoglobin extract of tilapia, which can be precipitated with ammonium sulfate. Mb molecular weight of tilapia is about 15.8 kDa, which can be collected by the treatment of ammonium sulfate with saturation between 60%-70% to remove unwanted protein. The absorption spectra show that Mb derivatives could be transferred to met-myoglobin after preliminary fractionating with ammonium sulfate.

Purification and Characterization ofα -l-Arabinopyranosidase andα -l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.