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Acta Biochimica et Biophysica Sinica Advance Access originally published online on April 15, 2009
Acta Biochimica et Biophysica Sinica 2009 41(5):399-406; doi:10.1093/abbs/gmp026
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© The Author 2009. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes

Driss Mountassif1,*, Tarik Baibai2,*, Latifa Fourrat2, Adnane Moutaouakkil3, Abdelghani Iddar3, M'Hammed Saïd El Kebbaj1 and Abdelaziz Soukri2

1 Laboratoire de Biochimie et Biologie Moléculaire, Université Hassan II-Aïn Chock, Faculté des Sciences Aïn Chock, km 8 route d'El Jadida BP. 5366, Mâarif, Casablanca, Morocco
2 Laboratoire de Physiologie et Génétique Moléculaire, Université Hassan II – Aïn Chock, Faculté des Sciences Aïn Chock, km 8 route d'El Jadida BP. 5366, Mâarif, Casablanca, Morocco
3 Unité de Radio-Immuno-Analyse, Département des Applications aux Sciences du Vivant, Centre National de l'Energie, des Sciences et des Techniques Nucléaires, BP 1382 RP, 10001 Rabat, Morocco

* Corresponding address. Tel: +212-22-230680/84; Fax: +212-22-230674; E-mail: baiman21{at}yahoo.fr (T.B.) & drissmountassif{at}yahoo.fr (D.M.)


  Abstract

A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12 [EC] ) from human erythrocytes. The comparison between this rapid method (one step) and the traditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of ~150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide (NAD+). Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43°C and 8.5, respectively. The kinetic parameters were also calculated: the Vmax was 4.3 U/mg and the Km values against G3P and NAD+ were 20.7 and 17.8 µM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.

Keywords    glyceraldehyde-3-phosphate dehydrogenase; immunoaffinity purification; human erythrocytes; characterization

Received: November 18, 2008; Accepted: January 5, 2009
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