Photosynthesis Research

, Volume 37, Issue 1, pp 81–86 | Cite as

A facile method to determine the CO2/O2 specificity factor for ribulose bisphosphate carboxylase/oxygenase

  • Garrett J. Lee
  • Roumen V. Kostov
  • Bruce A. McFadden
Technical Communication


A rapid method to determine the CO2/O2 specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO2 and O2 fixation at varying CO2/O2 ratios to determine the relative rates of each reaction. CO2 fixation is measured by the incorporation of the moles of14CO2 into 3-phosphoglycerate, while O2 fixation is determined by subtraction of the moles of CO2 fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined.

Key words

ribulose 1,5-bisphosphate assay rubisco specificity factor tau 



3-phosphoglyceric acid


N-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid)


1,5-bisphosphate carboxylase/oxygenase


d-ribulose 1,5-bisphosphate


d-xylulose 1,5-bisphosphate


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  1. Berhow MA, Saluja A and McFadden BA (1982) Rapid purification ofd-ribulose 1,5-bisphosphate carboxylase by vertical sedimentation in a reoriented gradient. Plant Sci Lett 27: 51–57.Google Scholar
  2. Chen PS, Toribara TY and Warner H (1956) Microdetermination of phosphorus. Anal Chem 28: 1756–1758.Google Scholar
  3. Edmondson DE, Kane HJ and Andrews TJ (1990) Substrate isomerization inhibits ribulosebisphosphate carboxylase-oxygenase during catalysis. FEBS Lett 260: 62–66.Google Scholar
  4. Harpel MR, Lee EH and Hartman FC (1993) Anion exchange analysis of ribulose bisphosphosphate carboxylase/oxygenase reactions: CO2/O2 specificity determination and identification of side products. Anal Biochem 209: 367–374.Google Scholar
  5. Horecker BL, Hurwitz J and Weissbach A (1958) Ribulose diphosphate. Biochem Prep 6: 83–90.Google Scholar
  6. Jordan DB and Ogren WL (1981) Species variation in the specificity of ribulose bisphosphate carboxylase/oxygenase. Nature 291: 513–515.Google Scholar
  7. Jordan DB and Ogren WL (1984) The CO2/O2 specificity of ribulose 1,5-bisphosphate carboxylase/oxygenase. Planta 161: 308–313.Google Scholar
  8. Kent SS and Tomany MJ (1984) Kinetic variance of ribulose-1,5-bisphosphate carboxylase/oxygenase from diverse taxonomic sources. Plant Physiol 75: 645–650.Google Scholar
  9. Laing WA and Ogren WL, Hageman RH (1974) Regulation of soybean net photosynthetic CO2 fixation by the interaction of CO2, O2, and ribulose1,5-diphosphate carboxylase. Plant Physiol 54: 678–685.Google Scholar
  10. Lee GJ and McFadden BA (1992) Serine-376 contributes to the binding of substrate by ribulose-bisphosphate carboxylase/oxygenase fromAnacystis nidulans. Biochemistry 31: 2304–2308.Google Scholar
  11. Parry MAJ, Keys AJ and Gutteridge S (1989) Variation in the specificity factor of C3 higher plant rubiscos determined by the total consumption of ribulose-P2. J Exp Bot 40: 317–320.Google Scholar
  12. Parry MAJ, Madgwick P, Parmer S, Cornelius MJ and Keys AJ (1992) Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity. Planta 187: 109–112.Google Scholar
  13. Purohit K, McFadden BA and Saluja A (1982) Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase. Anal Biochem 124: 158–166.Google Scholar
  14. Read BA and Tabita FR (1992) A hybrid ribulose bisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor. Biochemistry 31: 5553–5560.Google Scholar
  15. Storrø I and McFadden BA (1983) Ribulose bisphosphate carboxylase/oxygenase in toluene-permeabilizedRhodospirillum rubrum. Biochem J 212: 45–54.Google Scholar
  16. Wong C-H, Pollak A, McCurry SD, Sue JM, Knowles JR and Whitesides GM (1982) Synthesis of ribulose 1,5-bisphosphate: routes from glucose 6-phosphate (via 6-phosphogluconate) and from adenosine monophosphate (via ribose 5-phosphate). Meth in Enzymol 89: 108–121.Google Scholar
  17. Yokota A and Kitaoka S (1985) Correct pK values for dissociation constant of carbonic acid lower the reported Km values of ribulose bisphosphate carboxylase to half. Presentation of a nomograph and an equation for determining the pK values. Biochem Biophys Res Commun 131: 1075–1079.Google Scholar
  18. Zhu G, Jensen RG, Hallick RB and Wildner GF (1992) Simple determination of the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase by the specific radio-activity of [14C]glycerate 3-phosphate. Plant Physiol 98: 764–768.Google Scholar

Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Garrett J. Lee
    • 1
  • Roumen V. Kostov
    • 1
  • Bruce A. McFadden
    • 1
  1. 1.Department of Biochemistry and BiophysicsWashington State UniversityPullmanUSA

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