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Pakistan Journal of Biological Sciences
  Year: 2001 | Volume: 4 | Issue: 5 | Page No.: 564-568
DOI: 10.3923/pjbs.2001.564.568
 
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Purification and Characterization of Raphanin, A Neutral Protease, from Raphanus sativus Leaves

Sanaa T. El-Sayed

Abstract:
A neutral protease of 64000 Dalton molecular weight, named raphanin, has been isolated in a homogeneous state from leaves of Raphanus sativus (Radish). The enzyme was purified by a sequence of fractional precipitation with ammonium sulfate, adsorption on hydroxylapatite chromatography, followed by gel filtration chromatography on two successive columns of Sephadex G-120 and Sephadex G-200. The casein activity of the enzyme was tested in several steps of its purification and an increase of almost 168 fold was obtained. Raphanin has been purified to apparent homogeneity, as tested by gel filtration on Sephadex G-200 and showed a single protein band on polyacrylamide gel electrophoresis. Raphanin is active over a broad range of temperature (30-60°C) and has an optimum activity at 50°C. It has a maximum activity at pH 6.5-7.0 up to 120 min., when casein was used as substrate. Raphanin is highly heat stable for 60 min at 30°C with 23% loss of its activity only. The apparent Michael`s constants towards casein, haemoglobin, fibrin and collagen are 0.26, 0.44, 0.57 and 1.25% respectively. The enzyme was activated by Pb+2 and Cu+2 (46-55%) and inhibited by Hg+2, Ag+1 and EDTA (38-82%), but only moderately activated by Zn+2, p-chloromercuribenzoate and 2-mercaptoethanol (6-11%).
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How to cite this article:

Sanaa T. El-Sayed , 2001. Purification and Characterization of Raphanin, A Neutral Protease, from Raphanus sativus Leaves. Pakistan Journal of Biological Sciences, 4: 564-568.

DOI: 10.3923/pjbs.2001.564.568

URL: https://scialert.net/abstract/?doi=pjbs.2001.564.568

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