Protective Effect of Sundakai (Solanum torvum) Seed Protein (SP) Against Oxidative Membrane Damage in Human Erythrocytes.

Lipid peroxidation by ROS at the membrane level disturbs the inherit integrity of components activating subsequent alterations in the function. In this study, the protective effect of purified Sundakai (Solanum torvum) seed protein (SP) was tested against oxidative membrane damage in erythrocyte membrane. SP prevented oxidative RBC lysis induced by pro-oxidants; Fe:As (2:20 µmol), periodate (0.4 mM), and t-BOOH (1 mM) up to 86, 81, and 86 %, respectively. Further, SP prevented the Fe:As-induced K(+) leakage up to the tune of 95 %. The inhibition offered by SP on K(+) leakage was comparable to inhibition offered by quinine sulfate, a known K(+) channel blocker. SP dose dependently restored Na(+)K(+) ATPase and Ca(2+)Mg(2+) ATPase activities in erythrocyte membrane. The restoration of ATPase activity by SP was two times more than standard antioxidants BHA and α-tocopherol. Besides, SP at 1.6 µmol restored the membrane proteins over Fe:As induction when analyzed by SDS-PAGE, which was comparable to protection offered by BHA. In conclusion, SP is an effective antioxidant in preventing oxidative membrane damage and associated functions mediated by ROS. As SP is non-toxic, it can be used as an effective bioprotective antioxidant agent to cellular components.

PID15, a novel 6 kDa secreted peptide, mediates Naja naja venom phospholipase A2 induced apoptosis in isolated human peripheral lymphocytes.

BACKGROUND: Snake venoms are a complex mixture of active principles mainly peptides and proteins also including amino acids, nucleotides, free lipids, carbohydrates and metallic elements bound to proteins that interfere in several biological systems. In this study, we aimed to understand the mode of action of the apoptosis inducing ability of Naja naja venom phospholipase A2 (NV-PLA2) using isolated human peripheral lymphocytes. RESULTS: Human peripheral lymphocytes when incubated with Naja naja venom phospholipase A2 (NV-PLA2) induced up to 68% DNA fragmentation. The dialysed conditioned media obtained by incubating lymphocytes with NV-PLA2 at 15th min induced 44% DNA fragmentation, referred to as cmlp-active. Cmlp-active showed 20.5% increased protein concentration than the corresponding control condition media cmlp-c-15. Test for creatine kinase activity in cmlp-active proved negative and negligible amount of lactate dehydrogenase did not show significant DNA fragmentation. Fractionation of cmlp-active on Sephadex G-25 showed two peaks, major peak induced 38% DNA fragmentation, which was further rechromatographed on Sephadex G-25. The single peak obtained was named PID15 (Phospholipase A2Induced DNA fragmentation factor secreted at 15th min). Q-Tof MS/MS analysis of PID-15 showed it is a 6 kDa peptide. PID15 sequence analysis gave 40 amino acids in the following order, msilpcknvs iwvikdtaas dkevvlgsdr aikflylatg. The homology search for the sequence revealed it to be an Apoptosis Inducing Factor (AIF). CONCLUSION: Results indicate that the secretion of PID15 is dependent on concentration of NV-PLA2 treatment, incubation time and also on temperature and the probable membrane origin of PID15 and not of cytosolic origin with apoptosis inducing ability.

Purification and characterization of approximately 35 kDa antioxidant protein from curry leaves (Murraya koenigii L.).

Curry leaves (Murraya koenigii L. Spreng, Rutaceae) is an herbal species used in traditional medicine in eastern Asia. In this study, the antioxidant protein was purified by homogenization of curry leaves powder in Tris buffer, 65% ammonium sulphate precipitation and gel filtration on Sephadex G-75 column which resulted in three peaks (PI, PII and PIII). PII protein inhibited lipid peroxidation in human RBC ghost at 100 microg by 80%, whereas PI and PIII at 600 microg showed moderate antioxidant activity (40-50%). Homogeneity of PII was confirmed by rechromatographing on Sephadex G-75 and reverse phase HPLC. The antioxidant protein (PII) from curry leaves (APC) showed apparent molecular weight of 35 kDa by SDS-PAGE and MALDI/MS analysis. The protein nature of APC was established by the presence of amino acids and loss of antioxidant activity on heat and protease treatment. The APC at 0.8 microM inhibited lipoxygenase activity by 71%, effectively prevented diene, triene and tetraene lipids formation at 3 microM, and scavenged about 85% hydroxyl and DPPH radicals at 150-fold lesser concentration compared to BHA and alpha-tocopherol (400 microM). In addition, APC reduced cytochrome c and ferric ion, chelated ferrous ion, and inhibited ferrous sulfate: ascorbate-induced fragmentation and sugar oxidation to 80-90%. Thus, the present study demonstrates the in vitro characterization of antioxidant protein from the curry leaves (M. koenigii L.).

Purification and characterization of a approximately 34 kDa antioxidant protein (beta-turmerin) from turmeric (Curcuma longa) waste grits.

Beta-turmerin from turmeric (Curcuma longa) waste grits obtained after extraction of curcumin was purified by successive gel permeation chromatography. Homogeneity of beta-turmerin was confirmed by its movement as single band both in SDS-PAGE and as well as in native (basic) PAGE. The apparent molecular mass is approximately 34 kDa by SDS-PAGE. It is more hydrophobic protein and showed sharp single peak in RP-HPLC with retention time of 62.17 min. It is a glycoprotein as it shows the presence of amino sugars up to 0.021 gm%. In three different model systems i.e., linolenic acid micelles, erythrocyte membrane systems and liposomes, beta-turmerin at 0.125 microM offered 70%, 64%, and 60% inhibition of lipid peroxidation, which is 3200 times more efficient than the standard antioxidants BHA (400 microM) and alpha-tocopherol (400 microM). beta-turmerin inhibited diene-triene and tetraene conjugation up to 54%, 72% and 47%, respectively. beta-turmerin also effectively scavenges hydroxyl radicals when compared to BHA and alpha-tocopherol. beta-turmerin (2.5 microM) further inhibited the activation of PMNL mediated by fMLP up to the extent of 75%, where as standards BHA (400 microM) and mannitol (10 microM) inhibited the same to 65% and 55%, respectively. At 0.125 microM dose beta-turmerin prevented t-BOOH induced cell death at all time intervals. In addition to the above properties, it is non-toxic to lymphocytes as it did not affect the viability of cells. The mechanism of antioxidant action of beta-turmerin could probably be by counteracting/quenching of reactive oxygen species (ROS). We report the purification and characterization of beta-turmerin ( approximately 34 kDa), a potent antioxidant protein from turmeric waste grits.

New biological activity against phospholipase A2 by Turmerin, a protein from Curcuma longa L.

Turmerin is a protein from Turmeric (Curcuma longa L.) with a relative molecular mass of 14 kDa. The protein inhibits the enzymatic activity and neutralizes the pharmacological properties, such as cytotoxicity, oedema and myotoxicity of multitoxic phospholipase A2 (NV-PLA2) of cobra (Naja naja) venom at a 1:2.5 molar ratio of NV-PLA2:Turmerin. A Lineweaver-Burk plot indicates that Turmerin follows a linear mixed type of inhibition.