Purification and some properties of human placental glucose-6-phosphate dehydrogenase

Aksoy Y., Ogus I., Ozer N.

PROTEIN EXPRESSION AND PURIFICATION, vol.21, no.2, pp.286-292, 2001 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 21 Issue: 2
  • Publication Date: 2001
  • Doi Number: 10.1006/prep.2000.1370
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.286-292
  • Keywords: human placenta, glucose-6-phosphate dehydrogenase, purification, K-m values, activation energy, activation enthalpy, temperature activation factor (Q(10)), HUMAN-ERYTHROCYTE GLUCOSE-6-PHOSPHATE-DEHYDROGENASE, KINETIC-PROPERTIES
  • Hacettepe University Affiliated: Yes


Glucose-6-phosphate dehydrogenase was purified from human placenta using DEAE-Sepharose fast flow, 2',5'-ADP Sepharose 4B column chromatography, and chromatofocusing on PBE 94 with PB 74, The enzyme was purified with 62% yield and had a specific activity of 87 units per milligram protein. The pH optimum was determined to be 7.8, using zero buffer extrapolation method. The purified placental glucose-6-phosphate dehydrogenase gave two activity bands on native PAGE: one band, constituting about 3-5% of total activity, moved slower than the remaining 95%. Among the activity bands only the faster moving band gave a band on protein staining. The slower moving band, which probably corresponded to the higher polymeric form of the G6PD with high specific activity, was not seen on native PAGE due to insufficient protein for Coomassie brillant blue staining. The observation of one band on SDS-PAGE with an M-r of 54 kDa and a specific activity lower than expected, suggests the presence of both forms of the G6PD, the high polymeric form at low concentration and the inactive form at high concentration, in our preparation. Measuring the activities of placental glucose g-phosphate dehydrogenase between 20 and 50 degreesC, the activation energy, activation enthalpy, and Q(10) were calculated to be 8.16 kcal/mol, 7.55 kcal/mol, and 1.57, respectively. It was found that human placental G6PD obeys Michaelis-Menten kinetics. K-m values were determined using the concentration ranges of 20-300 muM for G6P and 10-200 muM for NADP(+). The K-m value for G6P was 40 muM the K-m value NADP(+) was found to be 20 muM. Double-reciprocal plots of 1/Vm vs 1/G6P (at constant [NADP(+)) and of 1/Vm vs 1/NADP(+) (at constant [G6P]) intersected at the same point on the 1/V-m, axis to give V-m = 87 U/mg protein. (C) 2001 Academic Press