Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase

K. Purohit, B. A. McFadden, Ashok Saluja

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Complete stoichiometry of the reaction catalyzed by ribulose 1,5-bisphosphate (RuBP) oxygenase from spinach and Rhodospirillum rubrum has been determined. Before initiation and after termination, RuBP has been measured either by release of equimolar orthophosphate at 25°C in the presence of 1 n NaOH or by complete carboxylation using 14CO2 and RuBP carboxylase. The RuBP-dependent oxygen consumption has been measured continuously with an oxygen electrode. After termination of catalysis, 3-phosphoglycerate production has been determined spectrophotometrically using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, α-glycerophosphate dehydrogenase, ATP, and NADH. To measure phosphoglycolate, this product was first hydrolyzed with alkaline phosphatase and the resultant glycolate oxidized by glycolate oxidase. Attendant H2O2 formation catalyzed by peroxidase has then been measured colorimetrically. Interference by ribulose in the measurement of glycolate can be easily corrected. Procedures are rapid and do not require separation of reactants and products. Results are in excellent accord with the expected stoichiometry for catalysis by RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase.

Original languageEnglish (US)
Pages (from-to)158-166
Number of pages9
JournalAnalytical Biochemistry
Volume124
Issue number1
DOIs
StatePublished - Jul 15 1982
Externally publishedYes

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Ribulose-Bisphosphate Carboxylase
Oxygenases
Stoichiometry
Assays
glycolic acid
Catalysis
Carboxylation
Triose-Phosphate Isomerase
Rhodospirillum rubrum
Oxygen
Glycerolphosphate Dehydrogenase
Glyceraldehyde-3-Phosphate Dehydrogenases
Spinacia oleracea
Oxygen Consumption
NAD
Peroxidase
Alkaline Phosphatase
ribulose-1,5 diphosphate
Electrodes
Adenosine Triphosphate

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase. / Purohit, K.; McFadden, B. A.; Saluja, Ashok.

In: Analytical Biochemistry, Vol. 124, No. 1, 15.07.1982, p. 158-166.

Research output: Contribution to journalArticle

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