Services on Demand
- Cited by SciELO
- Access statistics
On-line version ISSN 1414-431X
Braz J Med Biol Res vol. 31 n. 12 Ribeirão Preto Dec. 1998
Braz J Med Biol Res, December 1998, Volume 31(12) 1533-1535 (Short Communication)
Erythrocyte glucose-6-phosphate dehydrogenase activity assay and affinity for its substrate under "physiological" conditions
1Faculdade de Ciências Farmacêuticas, Universidade Estadual de Araraquara, Araraquara, SP, Brasil
2LIM 27, Instituto de Psiquiatria, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil
Correspondence and Footnotes
Glucose-6-phosphate dehydrogenase (G6PD) activity and the affinity for its substrate glucose-6-phosphate were investigated under conditions similar to the physiological environment in terms of ionic strength (I: 0.188), cation concentration, pH 7.34, and temperature (37oC). A 12.4, 10.4 and 21.4% decrease was observed in G6PD B, G6PD A+ and G6PD A- activities, respectively. A Km increase of 95.1, 94.4 and 95.4% was observed in G6PD B, G6PD A+ and G6PD A-, respectively, leading to a marked decrease in affinity. In conclusion, the observation of the reduced activity and affinity for its natural substrate reflects the actual pentose pathway rate. It also suggests a much lower NADPH generation, which is crucial mostly in G6PD-deficient individuals, whose NADPH availability is poor.
Key words: glucose-6-phosphate dehydrogenase, red blood cells, G6PD activity and affinity
Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) (EC 22.214.171.124) is a pentose shunt enzyme involved in the maintenance of adequate concentrations of reduced nicotinamide adenine dinucleotide phosphate (NADPH). This nucleotide, together with glutathione reductase, keeps glutathione in its reduced form (GSH), protecting the red cell against oxidative stress. There are two normal G6PD variants, G6PD B and G6PD A+, and other deficient polymorphic mutants such as G6PD A-, besides dozens of rare ones (1), some of which have been described by our group (2,3).
The standard methods used to assay G6PD activity and affinity (Michaelis-Menten constant - Km) for its substrate are currently performed using reaction conditions of 145 mOs and ionic strength I: 0.06, pH 8.0, at 37oC for activity assay and 25oC for Km determination. In the present study the enzyme activity as well as its affinity were determined under nearly physiological conditions regarding osmolarity (290 mOs) and ionic strength (I: 0:188), 3 mM MgCl2, pH 7.34, at 37oC. The activity assay was performed with NADP and glucose-6-phosphate (G6P) in excess. Under physiological conditions (37oC and 290 mOs) there was a 10-12% decrease in the activity assay, as well as an affinity decrease of 50% when compared to the standard methods (25oC and 145 mOs).
Blood was collected in the preservative solution ACD (citric acid, citrate and dextrose), and filtered through 1:1 microgranular cellulose:alpha-cellulose. The red cells were washed in buffered saline at 4oC and lysed and the enzymatic assays were carried out according to standard methods (4,5). The "physiological" reagent system was: 250 mM Tris-HCl, pH 7.34, 3 mM MgCl2, 0.2 mM NADP, which, together with the hemolysate and the substrate in excess (glucose-6-phosphate - 0.6 mM G6P) gave a final osmolarity of 290 mOs with I: 0.188 ionic strength at 37oC. G6PD purification and variant characterization were performed at 25oC according to standard methods (4,5) (activity against substrates, electrophoresis, Km for G6P and NADP, Ki for NADPH, pH curve, and thermal stability) and the Km was subsequently determined again under "physiological" conditions.
Other authors have focused on the effect of isolated factors on G6PD activity and affinity (6-9). In a different approach, the present study was planned to investigate how the activity and affinity would work in a nearly "physiological" environment involving several factors, a fact that impairs comparison of our data with those reported by others.
According to the results shown in Table 1, the studied variants showed different values in enzyme activity under" physiological" conditions (<10.4% in G6PD B, <12.4% in G6PD A+ and <21.5% in G6PD A-). The deficient variant G6PD A- appeared to be more sensitive to "physiological" conditions than the normal variants, a fact that, allied to its decreased activity, may enhance its difficulty in carrying out its metabolic functions, with impaired red cell performance.
The enzyme affinity for its physiological substrate glucose-6-phosphate was decreased when the "physiological" reagent system was employed, although the higher temperature used should theoretically facilitate the affinity. The ionic strength seems to play an important role in determining the affinity, impairing the access of the substrate to the active site of the enzyme.
In conclusion, the reduced activity of G6PD A-, taken together with its diminished affinity for its substrate, obtained under "physiological" conditions, in fact reflects the actual pentose shunt rate. This suggests a lower actual NADPH generation as well, which is crucial mostly in G6PD-deficient individuals, whose NADPH availability is poor.
1. Beutler E (1995). Glucose-6-phosphate dehydrogenase deficiency and other enzyme abnormalities. In: Beutler E, Lichtman MA, Coller BS & Kipps TJ (Editors), Williams Hematology. 5th edn. McGraw-Hill, New York, 564-581. [ Links ]
2. Barretto OC de O & Nonoyama K (1987). Gd(+) Cuiabá, a new rare glucose-6-phosphate dehydrogenase variant presenting normal activity. Human Genetics, 77: 201-202. [ Links ]
3. Barretto OC de O & Nonoyama K (1991). Gd(-) Carapicuiba, a rare glucose-6-phosphate dehydrogenase variant associated with moderate enzyme deficiency and chronic hemolysis. Brazilian Journal of Medical and Biological Research, 24: 133-139. [ Links ]
4. World Health Organization Scientific Group (1967). Standardization of procedures of the study of glucose-6-phosphate dehydrogenase. World Health Organization Technical Report Service, 366: 1-53. [ Links ]
5. Beutler E, Mathai CK & Smith JE (1968). Biochemical variants of glucose-6-phosphate dehydrogenase giving rise to congenital nonspherocytic hemolytic disease. Blood, 31: 131-150. [ Links ]
6. Cohen P & Rosemeyer MA (1969). Human glucose-6-phosphate dehydrogenase: Purification of the erythrocyte enzyme and the influence of ions on its activity. European Journal of Biochemistry, 8: 1-7. [ Links ]
7. Luzzatto L (1973). Studies of polymorphic traits for the characterization of populations: African populations South of the Sahara. Israel Journal of Medical Sciences, 9: 1181-1194. [ Links ]
8. Babalola AOG, Beetlestone JG & Luzzatto L (1976). Genetic variants of human erythrocyte glucose-6-phosphate dehydrogenase. Kinetic and thermodynamic parameters of variants A, B and A- in relation to quaternary structure. Journal of Biological Chemistry, 251: 2993-3002. [ Links ]
9. Yoshida A (1980). A contradiction between in vivo and in vitro activities of normal and variant glucose-6-phosphate dehydrogenase. Hemoglobin, 4: 769-780. [ Links ]
Address for correspondence: O.C. de O. Barretto, Av. Pedroso de Morais, 70, Apto. 101, 05420-000 São Paulo, SP, Brasil. Fax: +55-11-853-5894.
Presented at the XIII Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, Brasil, August 26-29, 1998. Research supported by FAPESP and CAPES. Received April 9, 1998. Accepted October 8, 1998.