Estimation of Molecular Size and Molecular Weights of Biological Compounds by Gel Filtration

Andrews, P.

doi:10.1002/9780470110362.ch1

Cited by 547 publications

(55 citation statements)

References 184 publications

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“…The average molecular weight of L-aspargenase was determined by gel filtration chromatography [5,49]. UsingSephacryl S-200 column (1x100).…”

Section: Determination Of Molecular Weightmentioning

confidence: 99%

Anticancer Activity of L-Asparaginase Produced from Vigna Unguiculata

Moharib

2018

World Scientific Research

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The present study aimed to production of purified L-asparaginase from Vignaunguiculata. Different physiological parameters, such as pH, temperature and incubation period, were optimized for growth and maximum L-asparaginase production. The optimum parameters were 37°C, 30 min and pH 8.5. Maximum L-asparaginase was 886.4U/ml with a specific activity of 1140.7 U/ml (31 fold purification with 28 %yield) were obtained at optimum conditions. The purified L-asparaginase produced from Vignaunguiculata was used for characterization and general properties. The effect of pH and temperature on L-asparaginase activity as well as stability at different pH and temperature were determined. The optimum pH 8.5 and 37ºC temperature on L-asparaginase showed 100% residual activity. Stability of pH around 8.5 and temperature 70ºC showed 90 and 78 % residual activity at 30 and 60 min respectively. The Lasparaginase showed high stability at alkaline pH (pH 8.5) when incubated for up to 60h.The molecular weight of the produced L-asparaginase was close to 68.5 kDa. Cytotoxic activity of Lasparaginase was examined in vitro using four carcinoma cell lines. L-aspargenase has higher effective in growth inhibition against HEPG2 and HCT-116 but lower against HELLA and MCF7 carcinoma cell lines. The data show that L-aspargenase has a higher cytotoxic activity against HEPG2 and HCT116, revealed higher percentage of cell death, indicating antitumor properties, and demonstrate direct effect on cancer cell proliferation of HEPG2 and HCT116. Therefore, Vignaunguiculata was considered to be a suitable source for production of Lasparaginase has higher activity and good stability. Purified L-asparaginase obtained from Vignaunguiculata could be employed in drug chemotherapy and treatment of cancer.

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“…The average molecular weight of L-aspargenase was determined by gel filtration chromatography [5,49]. UsingSephacryl S-200 column (1x100).…”

Section: Determination Of Molecular Weightmentioning

confidence: 99%

Anticancer Activity of L-Asparaginase Produced from Vigna Unguiculata

Moharib

2018

World Scientific Research

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“…The column was calibrated with a series of protein standards using the same buffer and flow rates to obtain the relationship between the elution volume and hydrodynamic radius. The hydrodynamic radii of lysozyme (Parmar and Muschol, 2009), carbonic anhydrase (Potschka, 1987), ovalbumin (Andrews, 1970), BSA (Andrews, 1970), b-casein (Marchesseau et al, 2002), and glycogen phosphorylase (DeVincenzi and Hedrick, 1967) were taken from the literature.…”

Section: Measurement Of Hydrodynamic Radiusmentioning

confidence: 99%

The Importance of Size and Disorder in the Cryoprotective Effects of Dehydrins

et al. 2013

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Dehydrins protect plant proteins and membranes from damage during drought and cold. Vitis riparia K 2 is a 48-residue protein that can protect lactate dehydrogenase from freeze-thaw damage by preventing the aggregation and denaturation of the enzyme. To further elucidate its mechanism, we used a series of V. riparia K 2 concatemers (K 4 , K 6 , K 8 , and K 10 ) and natural dehydrins (V. riparia YSK 2 , 60 kilodalton peach dehydrin [PCA60], barley dehydrin5 [Dhn5], Thellungiella salsuginea dehydrin2 , and Opuntia streptacantha dehydrin1 ) to test the effect of the number of K-segments and dehydrin size on their ability to protect lactate dehydrogenase from freeze-thaw damage. The results show that the larger the hydrodynamic radius of the dehydrin, the more effective the cryoprotection. A similar trend is observed with polyethylene glycol, which would suggest that the protection is simply a nonspecific volume exclusion effect that can be manifested by any protein. However, structured proteins of a similar range of sizes did not show the same pattern and level of cryoprotection. Our results suggest that with respect to enzyme protection, dehydrins function primarily as molecular shields and that their intrinsic disorder is required for them to be an effective cryoprotectant. Lastly, we show that the cryoprotection by a dehydrin is not due to any antifreeze proteinlike activity, as has been reported previously.

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“…The apparent molecular mass of the native enzymes were determined according to the method of Andrews (1970) using Sephadex G-200 (1.13 Â 100 cm) pre-equilibrated with 0.025 M sodium phosphate buffer pH 7.0, at a flow rate of 10 ml/h. The column was calibrated using cytochrome-c (12.3 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (BSA) (66 kDa), alcohol dehydrogenase (150 kDa) and b-amylase (200 kDa).…”

Section: Molecular Weight Determinationmentioning

confidence: 99%

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

2011

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a b s t r a c tTwo carboxylesterases (ME-III and ME-IV) have been purified to apparent homogeneity from the seeds of Mucuna pruriens employing ammonium sulfate fractionation, cation exchange chromatography on CMcellulose, gel-permeation chromatography on Sephadex G-100 and preparative PAGE. The homogeneity of the purified preparations was confirmed by polyacrylamide gel electrophoresis (PAGE), gel-electrofocussing and SDS-PAGE. The molecular weights determined by gel-permeation chromatography on Sephadex G-200 were 20.89 kDa (ME-III) and 31.62 kDa (ME-IV). The molecular weights determined by SDS-PAGE both in the presence and absence of 2-mercaptoethanol were 21 kDa (ME-III) and 30.2 kDa (ME-IV) respectively, suggesting a monomeric structure for both the enzymes. The enzymes were found to have Stokes radius of 2.4 nm (ME-III) and 2.7 nm (ME-IV). The isoelectric pH values of the enzymes, ME-III and ME-IV, were 6.8 and 7.4, respectively. ME-III and ME-IV were classified as carboxylesterases employing PAGE in conjunction with substrate and inhibitor specificity. The K m of ME-III and ME-IV with 1-naphthyl acetate as substrate was 0.1 and 0.166 mM while with 1-naphthyl propionate as substrate the K m was 0.052 and 0.0454 mM, respectively. As the carbon chain length of the acyl group increased, the affinity of the substrate to the enzyme increased indicating hydrophobic nature of the acyl group binding site. The enzymes exhibited an optimum temperature of 45°C (ME-III) and 37°C (ME-IV), an optimum pH of 7.0 (ME-III) and 7.5 (ME-IV) and both the enzymes (ME-III and ME-IV) were stable up to 120 min at 35°C. Both the enzymes were inhibited by organophosphates (dichlorvos and phosphamidon), but resistant towards carbamates (carbaryl and eserine sulfate) and sulphydryl inhibitors (p-chloromercuricbenzoate, PCMB).

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Estimation of Molecular Size and Molecular Weights of Biological Compounds by Gel Filtration

Cited by 547 publications

References 184 publications

Anticancer Activity of L-Asparaginase Produced from Vigna Unguiculata

Anticancer Activity of L-Asparaginase Produced from Vigna Unguiculata

The Importance of Size and Disorder in the Cryoprotective Effects of Dehydrins

Carboxylesterases from the seeds of an underutilized legume, Mucuna pruriens; isolation, purification and characterization

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