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Stoichiometry in the assay of ribulose bisphosphate oxygenase and carboxylase
ANALYTICAL
BIOCHEMISTRY
124, 158-166 (1982)
Stoichiometry
in the Assay of Ribulose Bisphosphate Oxygenase and Carboxylase’
K. PUROHIT,’ Biochemistry/Biophysics
B. A. MCFADDEN,~ AND ASHOK SALUJA~
Program and Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-4630 Received February 16, 1982
Complete stoichiometry of the reaction catalyzed by ribulose l$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 “C02 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, Lu-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 HzO, formation catalyzed by peroxidase has then been measured calorimetrically. 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 stoichiometrv for catalysis bv RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase. --
Phosphoglycolate was detected in the products obtained during photosynthesis by Scenedesmus (l), and its occurrence was attributed to nonenzymatic oxygenolytic cleavage of D-ribulose 1,Sbisphosphate (RuBP)’ into 3-phospho-D-glycerate (3PGA), and phosphoglycolate (P-glycolate). More recently, however, it has been established that P-glycolate is formed by enzymatic cleavage of RuBP and that the reaction is catalyzed by the oxygenase activity of RuBP carboxylase, which results in ’ Supported in part by Grant GM 19,972 from the NIH. ’ Present address: Nabisco Brands, Wilton, Connecticut 06897. 3 To whom inquiries should be addressed. ’ Present address: Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, New York 14853. ‘Abbreviations used: RuBP, D-ribulose l,%bisphosphate; 3-PGA, 3-phospho-D-glycerate; P-glycolate, phosphoglycolate. 0003-2697/82/l
10158-09$02.00/O
Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form rescrvcd.
158
equimolar quantities of 3-PGA and P-glycolate (2,3). In assays of RuBP oxygenase the measurement of RuBP-dependent oxygen consumption is most conveniently done with an oxygen electrode. 3-Phosphoglycerate is occasionally measured using a coupled enzymatic assay (4). As no suitable and convenient method is available for the determination of P-glycolate, this compound is rarely determined. Although attempts have been made to correlate the RuBP-dependent oxygen consumption with 3-PGA production (5), these results can at best yield only an approximate stoichiometry of the products of the oxygenase reaction and may result in overestimation of P-glycolate formation. This is because CO2 (supplied as HCO;) and Mg2+ are needed for the activation (6,7) of both carboxylase and oxygenase activities of RuBP carboxylase/oxygenase, and the 3PGA estimated is therefore the product of
RIBULOSE
BISPHOSPHATE
OXYGENASE
both activities. Obviously, a direct measurement of P-glycolate is crucial if stoichiometry for oxygenase catalysis is to be obtained. It is possible to identify and even quantify the production of P-glycolate by using [U14C]- or 32P-labeled RuBP as substrate and resolving the products by ion-exchange chromatography (1). These methods are not suitable as routine laboratory procedures when a large number of samples are assayed. Alternatively, P-glycolate can be converted into glyoxylate by the combined actions of P-glycolate phosphatase and glycolate oxidase and the glyoxylate measured as the phenylhydrazone derivative (81, but the method is reported to give about 75% recovery of P-glycolate. We now report an enzymatic method for P-glycolate determination using commercially available or easily prepared enzymes. This procedure and other methodology described are suitable to establish stoichiometry for catalysis by RuBP oxygenase. MATERIALS
AND METHODS
Materials. RuBP, 3-PGA, P-glycolate (tricyclohexylamine salt), NADH, NADP, recrystallized o-dianisidine - 2HC1, and enzymes were purchased from Sigma Chemical Company. a-Glycerophosphate dehydrogenase was purchased from Calbiochem, NaH14C0, from Amersham/Searle, and glycolic acid, 2,7-dihydroxynaphthalene, and hydroxylapatite from Aldrich and Bio-Rad, respectively. All more common chemicals were of analytical reagent quality. The commercial enzymes were assayed when used essentially as described by vendors. The purity of RuBP was established enzymatically using purified barley RuBP carboxylase and measuring the total incorporation of NaH14C03 into acid stable products (9) after completion of the reaction. Phosphoglycolate - tricyclohexamine * 2H20 was dissolved in the presence of HCl and converted to the Na+ salt by chromatography on Dowex 50 in the Naf form. The
STOICHIOMETRY
159
aqueous stock solution was kept frozen at -20°C. spinach glycola~e oxidase. Partially pure enzyme was prepared by the method as described by Harris and Stern (10). The enzyme eluting from the hydroxylapatite column in a single peak was precipitated by 60% saturation with respect to (NH4)*S04 and centrifuged at 30,OOOg for 30 min at 0°C. The pellet was suspended in 3.1 M (NH&SO4 which had been adjusted to pH 8.3 with NH,OH and which contained 2 mM FMN. The specific activity of the enzyme preparation, when assayed by the method of Harris and Stern (10) using buffer saturated with air at 3O”C, was 4.4 mol min-’ mg-’ protein in the presence of 20 mM glycolate. The preparation did not show any oxygen consumption with 0.5 mM RuBP, 3-PGA, or P-glycolate. The enzyme preparation was stable for at least 3 months at 4’C in ( NH4)$04 suspension. RuBP carboxylase/oxygenase. The spinach and R. rubru~ enzymes were pu~fied essentially as described by Saluja and McFadden ( 11) as was the enzyme from barley ( 12). RuBP oxygenase assay. The oxygenase activities of the spinach and barley enzymes were assayed at 30°C by measuring oxygen consumption using a Gilson oxygraph (13) with and without RuBP. The assay buffer, which contained 50 mM bicine and 20 mM MgCl* * 6H20, adjusted to pH 8.3, was saturated with CO,-free air at 30°C for I h. The final volume was 1.9 ml and contained 0.17-0.34 mM RuBP. The reaction was initiated with RuBP carboxylase/oxygenase that had been activated with 10 mM NaHCO, and 20 mM Mg*+ for at least 1 h at 30°C. The reaction was allowed to proceed beyond the period of linearity of oxygen consumption, which was about 1 min. No oxygen consumption was observed in the absence of RuBP or enzymes. The reactions were terminated either by rapidly transferring an aliquot from the reaction chamber to glass centrifuge tubes and heating in a boilingwater bath for 3 min or by addition of 50
160
PUROHIT,
MC FADDEN.
~1 of 50% trichloroacetic acid. The tubes were then chilled in ice and centrifuged at lO,~Og for 10 min at 4°C. For mixtures that had been acidified, supernatants were neutralized to pH 7 with an aliquot of NaOH and analyzed. Alternatively, the clarified supernatants were stored at -20°C for the determinations of 3-PGA, P-glycolate, and RuBP. Phosphate determinations. Inorganic and organic phosphates were determined by the method of Ames and Dubin ( 14), and the absorbance was measured at 800 nm. At 800 nm the absorbance with 16 nmol Pi was 8.3% lower than the reported absorbance (14) at 820 nm. Other details are indicated in the text. 3-PGA determinations. 3-Phosphoglycerate was determined (15) using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, and a-glycerophosphate dehydrogenase in the presence of ATP, Mg”‘, and NADH with recognition that 2 mol of NADH are oxidized during the reduction of 1 mol of 3PGA to cY-glycerophosphate. Phosphoglycolate determination. Phosphoglycolate (O-60 nmol) in 0.9 ml of 0.05 M bicine buffer, pH 8.3, was dephosphorylated by incubation with 1 unit of Escherichia cola’alkaline phosphatase at 37°C for 1 h and the reaction was terminated by immersion in a boiling-water bath for 3 min. After chilling in ice, a-dianisidine and horseradish peroxidase were added to each tube (see Fig. 3) and the tubes incubated at 30°C for 5 min. The reaction was initiated by addition of 0.17 unit of spinach glycolate oxidase in a final volume of 1.0 ml. The absorbance was read at 436 nm after 20 min against a reagent blank. RESULTS Determination of &BP in the Presence of 3-PGA and P-Glycolate by Measurement of NaOH-Labile Phosphate
Commercial RuBP is generally available at a purity of 70 to 85% but deteriorates on
AND SALUJA
RuBP
3-PGA , 0
of Phasphogiycoiote
1. 20
40 Time
0
SO
(Min)
FIG. 1. Time course of release of Pi from RuBP, 3PGA, and P-glycolate by 1 N NaOH at 25°C. RuBP (200 nmol) was incubated in 2.0 ml of 1.0 N NaOH. At specified times 0.2-ml samples were removed and neutralized with 0.1 ml of 2.0 N HsSO+ The concentration of 3-PGA and P-glycolate was 0.150 mM and both were treated as was RuBP. Alkali-labile Pi was determined as described in Table 1. The Pi present in unhydrolyzed samples has been subtracted.
storage. A preparation of RuBP, claimed to be 87% pure when assayed spectrophotometrically on the basis of ?I-PGA produced by RuBP carboxylase (9,1 S), was tested for the release of orthophosphate (Pi) by 1 N NaOH at 25°C in 30 min. Under these conditions, the release of Pi from RuBP was less than one per molecule. Under the same conditions of alkaline hydrolysis, Pi was not liberated from 3-PGA and P-glycolate. Moreover, the presence of 3-PGA and/or P-glycolate at OS- to 5-fold molar ratios to RuBP did not alter the release of Pi from RuBP. Only one of the two phosphate groups of RuBP was released during alkaline hydrolysis (Fig. 1), and the value of less than one Pi released per molecule of RuBP was therefore indicative of the purity of the preparation. On this basis the preparation of RuBP used was 74% pure. The preparation contained 0.26 mol Pijmol RuBP. As shown in Fig. 1 the release of Pi from RuBP by 1.0 N NaOH was complete in 20 min. Moreover, under the same conditions no Pi was released from P-glycolate or 3PGA. The time dependence of alkali-labile Pi release from RuBP (Fig. 1) was done 4 days after an earlier analogous assay, and in the interim the RuBP content decreased
RIBULOSE
0
1 20
BISPHOSPHATE
t 40 Time
eo
OXYGENASE
1
(Min)
FIG. 2. Time course of release of Pi from RuBP, 3PGA, and P-glycolate by alkaline phosphatase at 37’C. The reaction mixture (1 .O ml) contained 50 mM bicine, pH 9.4, 10 mM Me, and 0.4 mM RuBP, P-glycolate, or 3-PGA in a final volume of 1.O ml. After transferring 0.05 ml to an equal volume of 20% trichloroacetic acid (TCA), the reaction was initiated with 1 unit of calf intestinal alkaline phosphatase (1000 units/mg proteins) contained in 1 ~1 of water. At times indicated, 0.05-ml samples were transferred analogously to TCA. Aliquots of the WA-quenched samples were used for the Pi determination as described in the footnotes to Table 1. Due to the negligible quantity of protein added, no clarification of the samples was necessary.
from 80 to 74% and the Pi present increased from 0.20 to 0.26 mol/mol RuBP even though the preparation had been kept frozen at -20°C. From these results it is apparent that RuBP can be determined in the presence of both P-glycolate and 3-PGA. The liberation of only one phosphate group by alkali is in agreement with Paech et al. (16) but conflicts with the report of Horecker et al. (17) that both phosphate groups of RuBP are labile to alkali. The value of 2.8 min for the half-life of RuBP in 1 N NaOH reported by Horecker et al. (17) is close to the value obtained for the release of Pi from RuBP (Fig. 1) under identical conditions. Although P-glycolate and 3-PGA are refractory to alkaline hydrolysis at 25°C by 1 N NaOH, these compounds are quantitatively hydrolyzed by alkaline phosphatase (Fig. 2). Alkaline phosphatase released almost one equivalent of Pi per mole of P-glycolate or 3-PGA and accounted for the approximate 95% purity of these compounds. Both phosphate groups of RuBP were re-
161
STOICHIOMETRY
leased by alkaline phosphatase. The purity of a RuBP sample based on the hydrolysis of one of the two phosphate groups by alkali was found to be in perfect agreement with the release of two phosphate groups by alkaline phosphatase. In experiments where the hydrolyses with alkaline phosphatase were done with mixtures of RuBP, P-glycolate, and 3-PGA individually or in various combinations, the total Z’i liberated was equal to the sum of Pi liberated by individual components. Either calf intestinal or E. coli alkaline phosphatase could be used for the hydrolysis of RuBP, P-glycolate, or 3-PGA. The calf intestinal alkaline phosphatase was optimally active in the pH range of 9.4 to 10.4 in the presence of 1 mM Mg”, whereas the enzyme from E. coli yielded identical results between pH 8 and 8.6 and 1 and 20 mM Mg 2+. Similar results were also obtained with wheat germ acid phosphatase at pH 4.5 in the presence of 10 mM Mg’+. ~i~~~lties in the determination glycolate by Chemical Assay
of P-
The procedure described by Calkins (18) for the determination of glycolate by reaction with 2,7-dihydroxynapthalene in 33 N H2S04 in a boiling-water bath yielded about 6% of absorbance at 530 nm with P-glycolate as compared to an equivalent amount of glycolate. By conducting the above reaction at 121 “C in an autoclave for 15 min, a linear increase in the absorbance up to 200 nmol of P-glycolate was achieved. At this temperature, however, severe interference was caused by RuBP, Tris, Ammediol, EDTA, EGTA, or GSH, and slight interference was caused by 3-PGA. EDTA, EGTA, and Ammediol increased the intensity of absorbance without shifting the absorbance maxima, but the presence of RuBP completely masked the 535nm absorbance maxima and shifted them to 420 nm. Attempts to remove RuBP from a mixture containing RuBP, P-glycolate, and 3-PGA by reacting with dihydrox-
162
PUROHIT,
MC FADDEN.
yboryl-substituted cellulose ( 19) or by reacting with hydrazine-substituted resins were only partially successful or resulted in high dilution of P-glycolate, lowering its concentration almost beyond the sensitivity of the Calkins procedure. All attempts to eliminate the interference by the ribulose moiety using alkaline phosphatase-dephosphorylated synthetic reaction mixtures followed by the Calkins reaction at 100 or 121 “C yielded unsatisfactory results. Interference by the buffers could be eliminated by substituting bicine, Hepes, or TES buffers for Tris or Ammediol, as these buffers were unreactive at a concentration of 0.1 M in the sample assayed. The procedure of anion-change chromatography (1) was found to be of limited applicability for separation and routine assay of P-glycolate. Attempted Enzymatic Glycolate
Assays of P-
The assay of P-glycolate by dephosphorylation with alkaline phosphatase followed by rephosphorylation in the presence of pyruvate kinase, (Mg*+, K+) ATP, and phosphoenolpyruvate followed by estimation of the resulting pyruvate by lactate dehydrogenase and NADH seemed feasible. Assays were therefore attempted at several pH values from 7 to 8.3. Although glycolate-dependent oxidation of NADH was observed in the presence of 0.5 mM glycolate, the rate was too low to measure at 50 PM glycolate. Attempts to phosphorylate ADP with the donor P-glycolate in the presence of an excess of pyruvate kinase with parallel estimation of ATP using glucose, hexokinase, and glucosed-phosphate dehydrogenase was feasible at high P-glycolate concentrations and pH 8.3, but the reaction rate at 50 pM P-glycolate was too slow to be of use. Taking advantage of catalysis by lactate dehydrogenase of glycolate oxidation, we tried to pull the reaction toward glyoxylate in the presence of semicarbazide at pH 6.5 to 8.5. Although nonenzymatic glyoxylate
AND SALUJA
semicarbazone formation at pH 6 with known quantities of glyoxylate was quantitative, the coupled reaction rate with 50 pM glycolate was extremely slow. Attempted Determination of Glycolate Resulting from Dephosphorylation of PGlycolate as Glyoxylate Phenylhydrazone The oxidation of glycolate by glycolate oxidase results in the formation of H202 and glyoxylate (20), which may be further oxidized to CO*, H20, and formate by H202. All attempts to trap glyoxylate produced as the glyoxylate phenylhydrazone by the method of Baker and Tolbert (2 1) gave variable recoveries of glycolate derived from Pglycolate, even in the presence of 150 to 4000 units of catalase. However, the production of glyoxylate phenylhydrazone from authentic glyoxylate was quantitative as measured by the increase in absorbance at 324 nm. This reaction with glyoxylate was not influenced by the presence of alkaline phosphatase, catalase, and glycolate oxidase individually or in combination. In these experiments glycolate was generated by complete dephosphorylation of the tricyclohexylamine salt of phosphoglycolate by alkaline phosphatase. It is possible that cyclohexylamine might have been the cause of our unsatisfactory results, but this aspect was not investigated. Determination of Glycolate by Oxidation with Spinach Glycolate Oxidase and Measurement of the Resultant H202 The absorbance at 436 nm due to the chromophore resulting from the reaction of H202, generated stoichiometrically through catalysis by glycolate oxidase, with a-dianisidine in the presence of peroxidase obeyed Beer’s law for up to 60 nmol of authentic glycolate. Similar results were obtained for glycolate arising from complete hydrolysis of tricyclohexyl ammonium P-glycolate by alkaline phosphatase, but the absorbance
RIBULOSE
Glycolote
BISPHOSPHATE
OXYGENASE
(nmoles)
FIG. 3. Standard curve for glycolate. Sodium P-glycolate. was dephosphorylated in 1.0 ml of 0.05 M bicine (pH 8.3) by 1 unit of E. coli alkaline phosphatase for 1 h at 37°C. The tubes were heated in a boiling-water bath for 3 min, chilled in ice, and stirred briefly with a Vortex mixer. Aliquots of 0.9 ml containing the quantities specified were used for the determinations. To each tube, 0.085 ml of aqueous solution of 1.755 mM a-dianisidine 2 HCl* 2 Hz0 and 4.56 purpuroglin units of horseradish peroxidase in 0.015 ml of 0.1 M bicine buffer, pH 8.3, was added. After equilibration at 30°C for 5 min, the reaction was initiated by adding 5.0 ~1 containing 0.17 units of spinach glycolate oxidase. The absorbance was read at 436 nm in a Cary 14 spectrophotometer, after incubation for 20 min at 3O’C with correction for a reagent blank. In some experiments 100-400 nmol of 3-PGA was also present in the original alkaline phosphatase-treated mixture.
was about 20% lower than that obtained with authentic glycolic acid. This disparity disappeared when P-glycolate was used as the Na’ salt prior to dephosphorylation with alkaline phosphatase. The development of color was complete within 20 min at 3O”C, and further incubation up to 40 min did not alter the absorbance readings in the presence of 0, 5, or 20 mM Mg2+. Dephosphorylation of P-glycolate is a prerequisite of any method for glycolate determination using glycolate oxidase. In Fig. 2 it is shown that both 3-PGA and RuBP are also dephosphorylated by alkaline phosphatase. Thus, the enzymatically dephosphorylated reaction mixture contains variable amounts of ribulose, glycerate, and glycolate. In the absence of any information on the reactivity of ribulose and glyceric acid with glycolate oxidase and horseradish peroxidase, the interference by glyceric acid and
STOICHIOMETRY
163
ribulose in the glycolate assay was investigated. The absorbance at 436 nm produced by glycolate arising from P-glycolate is shown in Fig. 3. The slope obtained was in perfect agreement with the molar absorption coefficient of 8.3 X lo3 M cm-’ for the chromophore generated by the reaction of H202 with a-dianisidine in the presence of peroxidase (22) and was unaffected by the presence of 100 to 400 nmol of 3-PGA in the original mixture. Unlike glycerate, ribulose generated by alkaline phosphatase from RuBP increased the intercept values of the plots obtained with varying glycolate in the glycolate assay system (Fig. 4). The increase in the intercept values, however, was proportional to the RuBP originally present (see inset, Fig. 4), and no alteration of the slopes was noticed. A linear least-squares regression analysis of the absorbance at 436 nm in the presence
FIG. 4. Interference of ribulose in the determination of glycolate. From 9.4 to 56.4 nmol P-glycolate alone (8) or in the presence of 85 (O), 425 (A), and 850 (8) nmol RuBP (corrected for purity by enzymatic assay or by alkaline hydrolysis) were dephosphoryiated for 1 h at 37°C by 1 unit of E. coli alkaline phosphatase in 0.9 ml of 0.05 M bicine buffer, pH 8.3, containing 5 mM Mg’+. After heat quenching, the entire reaction mixture was used for color development as described in Fig. 3. Each curve was generated by linear least-squares regression analysis. The inset shows a plot of the y intercept vs concentration of ribulose bisphosphate (RuBP).
164
PUROHIT,
MC FADDEN, TABLE
STOICHIOMETRY
FOR
AND SALUJA 1
RuBP OXYGENASE
Experiment number
Enzyme source
O2 consumed (nmol)
Phosphoglycolate produced” (nmol)
1 2 3
Spinach Spinach Barley
127 223 172
135 240 213
Phosphoglycolate: oxygen 1.06 1.08 1.24
Note. The reactions were interrupted after 6 to 8 min of incubation and an aliquot portion rapidly transferred to a boiling-water bath. Thus, the period of phosphoglycolate production slightly exceeded the period of oxygen consumption. ’ Phosphoglycolate was measured by correcting the OD4s6 for absorbance derived from RuBP (Fig. 4). Residual RuBP was determined from the amount of alkali-labile phosphate remaining. When residual RuBP was measured by using H’%ZO; and running the RuBP carboxylase reaction to completion, the phosphoglycolate:02 ratios for experiments 1 to 3 were 1.07, 1.10, and 1.22, respectively.
of 0, 85, 425, and 850 nmol of ribulose with glycolate varying from 9.4 to 56.4 nmol/ml of the assay mixture yielded a mean slope of 0.008 + 0.0003, the uncertainty being at the level of 1 standard deviation. This value agreed closely with that obtained in the presence or absence of glycerate (Fig. 3).
Stoichiometry Oxygenase
Experiment number and enzyme sourcea (1) (2) (3) (4) (5) (6) (7)
Spinach minus RuBP control Minus enzyme control Spinach Spinach Spinach Spinach Spinach
(8) R rubrum
(9) Minus enzyme
control
STOICHIOMETRY
O2 consumed (nmol) 0 0 74 80 167 230 80 94 0
2 FOR
Phosphoglycolate produced* (nmol) 0 0 84 82 160 240 76 96 0
by RuBP
In Table 1, data establish that the measured P-glycolate:02 ratio was close to 1. This ratio slightly exceeded 1 because of the methodology used to stop both reactions. Of
TABLE COMPLETE
for Catalysis
RuBP OXYGENASE 3-PGA pdUWd (nmol)
Residual RuBP (nmol)
0 0 135 160 310 596 186 219 0
0 777 712 1530 590 1150 1475 1411 1528
Theoretical residual RuBP’ (nmol)
Phosphoglycolate: oxygen -
800 693 1479 563 1184 1468 1143 1600
1.14 1.03 0.96 1.04 0.95 1.02 -
o Reactions were quenched by the addition of acid. Incubations were from 1.1 to 7.5 min with 50 to 300 pg spinach enzyme added except in Exp. 2. In Exp. 8, 200 wg of R. rrtbrum enzyme were added. In Exp. 1, RuBP was not added to an otherwise complete reaction mixture containing 50 ug of spinach enzyme. to correct for residual RuBP. b Calculated from ODus as described for Table 1 using the enzymatic assay (with H”C0;) c Initial RuBP in Exps. 2, 3, and 5 was 800 nmol and in Exps. 4 and 6-9 was 1600 nmol. ’ Stoichiometrics for catalysis by RuBP carboxylase and oxygenasc are RuBP+ RuBP-’
+ COa + HsO * + O1 -+ 3-PGA-’
2(3-PGA-‘) + 2 H+ + P-glycolate-’ + 2 H+
Theoretical residual RuBP was calculated by (i) averaging oxygen consumed with P-glycolate produced to yield RuBP disappearing via RuBP oxygenase (RuBP,,); (ii) subtracting RuBP.., from 3-PGA total to yield 3-PGA appearing via RuBP carboxylase (3-PGA&; (iii) halving 3-PG&.to yield RuBP disappearing via RuBP carboxylase (RttBP..,); and (iv) theoretical residual RuBP = RuBPr,,ru., - (RuBPee, + RuBP,,).
RIBULOSE
BISPHOSPHATE
OXYGENASE
particular interest is the fact that ODdj6 values can be corrected for contributions due to unreacted RuBP measured either enzymatically or chemically by release of Pi by alkali. The latter methods were in excellent agreement (Table 1). In Table 2, data establish that the measured P-glycolate:02 ratio was very close to 1 when the RuBP oxygenase reaction was terminated with acid. Measured values of residual RuBP were in excellent agreement with theoretical values for residual RuBP when a correction was made for RuBP utilization by the simultaneous competing reaction catalyzed by RuBP carboxylase (Table 2). Analysis of the data in Table 2 reveals that the mole ratio of RuBP disappearing via RuBP oxygenase to that disappearing via RuBP carboxylase is 1.4-3.1 under the experimental conditions employed. DISCUSSION
A frequently used assay for the oxygenase activity of RuBP carboxylase simply involves the measurement of RuBP-dependent O2 consumption. This method is not fully adequate, however, because reaction products are not measured. Although a good enzymatic analysis of one of the products, 3PGA, exists, this compound is also produced by RuBP carboxylase. Unfortunately, prior to the present work there was not a good assay available for the second, and unique product, P-glycolate. We have described here such an assay; in addition to enabling facile measurement of the stoichiometry for the RuBP oxygenase reaction, this assay yields results in excellent agreement with those of Lorimer and colleagues (2,3). After termination of oxygen consumption, residual RuBP, P-glycolate, and 3-PGA can be measured in separate aliquot portions of a single reaction mixture. The results not only specifically measure the stoichiometry for catalysis by RuBP oxygenase but also allow a crude estimate of RuBP oxygenase:
STOICHIOMETRY
165
carboxylase ratio under the conditions employed. For example, these ratios varied between 1.4 and 3.1 in the presently described research. It is crucial to note, however, that bicarbonate was used at a sufficient concentration to activate fully both RuBP oxygenase and carboxylase, yet was diluted 19- to 30-fold to a final concentration of 6 pM CO2 to minimize inhibition of the former activity by COZ. Thus, under these conditions the concentration of CO* severely limited the rate of the RuBP carboxylase reaction. Clearly, it is impossible to obtain near maximal velocities for both oxygenase and carboxylase when measured simultaneously (23,24).
The ratio of RuBP oxygenase:carboxylase for the spinach enzyme is normally around 0.13 when the oxygenase is measured at 250 PM 0, under otherwise optimal conditions (25,26). The present methodology could be easily modified to measure RuBP carboxylase under optimal conditions. For example, after the enzymatic assay of 3-PGA, the addition of saturating HCO; should yield a rate of NADH oxidation that is maximal and proportional to RuBP carboxylase, providing residual RuBP is saturating and all other components in the coupled enzyme assay mixture are in excess. It is shown that RuBP can be determined quantitatively using RuBP carboxylase and measuring the incorporation of H14CO; incorporation into 3-PGA. Results are in excellent agreement with those yielded by decomposition of RuBP by alkali. Both assays are specific and other components in the reaction mixture containing RuBP carboxylase/oxygenase do not interfere. An elegant alternative for measuring the stoichiometry for the RuBP oxygenase reaction has recently been described (23). This method is based upon use of [ 1-3H]RuBP and measurement of oxygen uptake and of [ 3H]P-glycolate production after separation by anion-exchange chromatography. Use of H14CO; to activate the oxygenase also enables the determination of [ 14C]P-glycerate,
166
PUROHIT,
MC FADDEN,
which is a measure of the competing carboxylase reaction. Initial and residual RuBP are also measured, as is total 3-PGA produced. The resultant stoichiometry is good, but the method is cumbersome in that [l3H]Ru13P must be prepared and P-glycolate must be isolated. The methods described enable reliable assays for RuBP oxygenase. Although each study of oxygen ~nsumption requires an oxygen electrode, durations are short and up to 20 rates/h can be measured. Many subsequent assays of reactant stoichiometry can be simuitaneously performed. Thus, the methodology enables a large number of assays per day. We recommend that iaboratories use this technique to randomly check the reliability of faster assays in which RuBP-de~ndent oxygen consumption is measured. ACKNOWLEDGMENT Much of this man~c~pt was completed at the Technical University of Munich (TUM), Freising-Weihenstephan, where B.A.M. was a Humboldt Senior Scientist. He thanks Berthold Hock for the many courtesies extended while a guest investigator at the TUM.
REFERENCES 1. Goodman, M., Benson, A. A., and Calvin, M. (1955) J. Amer. Chem. Sot. 20,4257-4261. 2. Andrews, T. J., Lorimer, G. H., and Tolbert, N. E. (1973) Bi~hemjstry 12, 11-18. 3. Lorimer, G. H., Andrews, T. J., and Tolbert, N. E. (1973) Biochemistry 12, 18-23. 4. Jakoby, W. B., Brummond, D. O., and Ochoa, S. (1956) J. Biol. Chem. 218,811~822. 5. McFadden, B. A., Lord, J. M., Rowe, A., and Dilks, S. (1975) Eur. J. B&hem. 54, 195-206. 6. Lorimer, G. H., Badger, M. R., and Andrews, T. J. (1976) Biochemistry 15, 529-536.
AND SALUJA
7. Badger, M. R., and Lorimer, G. H. (1976) Arch. Biochem. Biophys. 145,723-729. 8. Bowes, G., Ogren, W. L., and Hageman, R. H. (1971) Biochem. Biophys. Res. Commun. 45, 716-722. 9. McFadden, B. A., Tab&a, F. R., and Kuehn, G. D. (1975) in Methods in Enzymology (Wood, W. A., ed.), Vol. 42, pp. 461-472, Academic Press, New York. 10. Harris, G. C., and Stern, A. I. (1978). J. Exp. Bot. 29,561-566. 11. Saluja, A., and McFadden, B. A. (1982) Biochemistry 21, 89-95. 12. Saluja, A., and McFadden, B. A. (1978) FEBS Lett. %, 361-363. 13. Lorimer, G. H., Badger, M. R., and Andrews, T. J. (1977) Anal. Bioehem. 78,66-75. 14. Ames, B. N., and Dubin, D. T. ( 1960) J. Biof. Chem. 235, 769-777. 15. Czok, R., and Eckert, L. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H., ed.), pp. 224-227, Academic Press, New York, 16. Paech, C., Pierce, J., McCurry, S. D., and Tolbert, N. E. (1978) Bioehem. Biophys. Res. Commun 83, 1084-1092. 17. Horecker, B. L., Hurwitz, J., and Weissbach, A. (1956) J. Biol. Chem. 218,785-794. 18. Calkins, V. P. (1943) ind. Erzg. Chem. 15,762-763. 19. Weith, H. L., Wiebers, J. L., and Gelham, P. T. (1970) Biochemistry 9, 4396-4401. 20. Zelitch, G. (1955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 1, pp. 528-532, Academic Press, New York. 21. Baker, A. L., and Tolbert, N. E. (1966) in Methods in Enzymology (Wood, W. A., ed.), Vol. 9, pp. 338-342, Academic Press, New York. 22. Berm, E., and Bergmeyer, H. (1965) in Methods of Enzymatic Analysis, (Bergmeyer, H., ed.), pp. 633-635, Academic Press, New ‘cork. 23. Jordan, D. B., and Osren, W. L. ( 198 1) Plant Physiol. 67, 237-245. 24. Chollet, R., and Anderson, L. L. (1976) Arch. Biothem. Biophys. 176, 344-351. 25. Lorimer, G. H., Badger, M. R., and Andrews, T. J. (1976) Biochemistry 15, 529-536. 26. Lorimer, G. H., Badger, M. R., and Andrews, T. J. (1977) Anal. Biochem. 78, 66-75.
BIOCHEMISTRY
124, 158-166 (1982)
Stoichiometry
in the Assay of Ribulose Bisphosphate Oxygenase and Carboxylase’
K. PUROHIT,’ Biochemistry/Biophysics
B. A. MCFADDEN,~ AND ASHOK SALUJA~
Program and Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-4630 Received February 16, 1982
Complete stoichiometry of the reaction catalyzed by ribulose l$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 “C02 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, Lu-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 HzO, formation catalyzed by peroxidase has then been measured calorimetrically. 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 stoichiometrv for catalysis bv RuBP oxygenase and also enable an estimate of competing catalysis by RuBP carboxylase. --
Phosphoglycolate was detected in the products obtained during photosynthesis by Scenedesmus (l), and its occurrence was attributed to nonenzymatic oxygenolytic cleavage of D-ribulose 1,Sbisphosphate (RuBP)’ into 3-phospho-D-glycerate (3PGA), and phosphoglycolate (P-glycolate). More recently, however, it has been established that P-glycolate is formed by enzymatic cleavage of RuBP and that the reaction is catalyzed by the oxygenase activity of RuBP carboxylase, which results in ’ Supported in part by Grant GM 19,972 from the NIH. ’ Present address: Nabisco Brands, Wilton, Connecticut 06897. 3 To whom inquiries should be addressed. ’ Present address: Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, New York 14853. ‘Abbreviations used: RuBP, D-ribulose l,%bisphosphate; 3-PGA, 3-phospho-D-glycerate; P-glycolate, phosphoglycolate. 0003-2697/82/l
10158-09$02.00/O
Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form rescrvcd.
158
equimolar quantities of 3-PGA and P-glycolate (2,3). In assays of RuBP oxygenase the measurement of RuBP-dependent oxygen consumption is most conveniently done with an oxygen electrode. 3-Phosphoglycerate is occasionally measured using a coupled enzymatic assay (4). As no suitable and convenient method is available for the determination of P-glycolate, this compound is rarely determined. Although attempts have been made to correlate the RuBP-dependent oxygen consumption with 3-PGA production (5), these results can at best yield only an approximate stoichiometry of the products of the oxygenase reaction and may result in overestimation of P-glycolate formation. This is because CO2 (supplied as HCO;) and Mg2+ are needed for the activation (6,7) of both carboxylase and oxygenase activities of RuBP carboxylase/oxygenase, and the 3PGA estimated is therefore the product of
RIBULOSE
BISPHOSPHATE
OXYGENASE
both activities. Obviously, a direct measurement of P-glycolate is crucial if stoichiometry for oxygenase catalysis is to be obtained. It is possible to identify and even quantify the production of P-glycolate by using [U14C]- or 32P-labeled RuBP as substrate and resolving the products by ion-exchange chromatography (1). These methods are not suitable as routine laboratory procedures when a large number of samples are assayed. Alternatively, P-glycolate can be converted into glyoxylate by the combined actions of P-glycolate phosphatase and glycolate oxidase and the glyoxylate measured as the phenylhydrazone derivative (81, but the method is reported to give about 75% recovery of P-glycolate. We now report an enzymatic method for P-glycolate determination using commercially available or easily prepared enzymes. This procedure and other methodology described are suitable to establish stoichiometry for catalysis by RuBP oxygenase. MATERIALS
AND METHODS
Materials. RuBP, 3-PGA, P-glycolate (tricyclohexylamine salt), NADH, NADP, recrystallized o-dianisidine - 2HC1, and enzymes were purchased from Sigma Chemical Company. a-Glycerophosphate dehydrogenase was purchased from Calbiochem, NaH14C0, from Amersham/Searle, and glycolic acid, 2,7-dihydroxynaphthalene, and hydroxylapatite from Aldrich and Bio-Rad, respectively. All more common chemicals were of analytical reagent quality. The commercial enzymes were assayed when used essentially as described by vendors. The purity of RuBP was established enzymatically using purified barley RuBP carboxylase and measuring the total incorporation of NaH14C03 into acid stable products (9) after completion of the reaction. Phosphoglycolate - tricyclohexamine * 2H20 was dissolved in the presence of HCl and converted to the Na+ salt by chromatography on Dowex 50 in the Naf form. The
STOICHIOMETRY
159
aqueous stock solution was kept frozen at -20°C. spinach glycola~e oxidase. Partially pure enzyme was prepared by the method as described by Harris and Stern (10). The enzyme eluting from the hydroxylapatite column in a single peak was precipitated by 60% saturation with respect to (NH4)*S04 and centrifuged at 30,OOOg for 30 min at 0°C. The pellet was suspended in 3.1 M (NH&SO4 which had been adjusted to pH 8.3 with NH,OH and which contained 2 mM FMN. The specific activity of the enzyme preparation, when assayed by the method of Harris and Stern (10) using buffer saturated with air at 3O”C, was 4.4 mol min-’ mg-’ protein in the presence of 20 mM glycolate. The preparation did not show any oxygen consumption with 0.5 mM RuBP, 3-PGA, or P-glycolate. The enzyme preparation was stable for at least 3 months at 4’C in ( NH4)$04 suspension. RuBP carboxylase/oxygenase. The spinach and R. rubru~ enzymes were pu~fied essentially as described by Saluja and McFadden ( 11) as was the enzyme from barley ( 12). RuBP oxygenase assay. The oxygenase activities of the spinach and barley enzymes were assayed at 30°C by measuring oxygen consumption using a Gilson oxygraph (13) with and without RuBP. The assay buffer, which contained 50 mM bicine and 20 mM MgCl* * 6H20, adjusted to pH 8.3, was saturated with CO,-free air at 30°C for I h. The final volume was 1.9 ml and contained 0.17-0.34 mM RuBP. The reaction was initiated with RuBP carboxylase/oxygenase that had been activated with 10 mM NaHCO, and 20 mM Mg*+ for at least 1 h at 30°C. The reaction was allowed to proceed beyond the period of linearity of oxygen consumption, which was about 1 min. No oxygen consumption was observed in the absence of RuBP or enzymes. The reactions were terminated either by rapidly transferring an aliquot from the reaction chamber to glass centrifuge tubes and heating in a boilingwater bath for 3 min or by addition of 50
160
PUROHIT,
MC FADDEN.
~1 of 50% trichloroacetic acid. The tubes were then chilled in ice and centrifuged at lO,~Og for 10 min at 4°C. For mixtures that had been acidified, supernatants were neutralized to pH 7 with an aliquot of NaOH and analyzed. Alternatively, the clarified supernatants were stored at -20°C for the determinations of 3-PGA, P-glycolate, and RuBP. Phosphate determinations. Inorganic and organic phosphates were determined by the method of Ames and Dubin ( 14), and the absorbance was measured at 800 nm. At 800 nm the absorbance with 16 nmol Pi was 8.3% lower than the reported absorbance (14) at 820 nm. Other details are indicated in the text. 3-PGA determinations. 3-Phosphoglycerate was determined (15) using phosphoglycerokinase, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase, and a-glycerophosphate dehydrogenase in the presence of ATP, Mg”‘, and NADH with recognition that 2 mol of NADH are oxidized during the reduction of 1 mol of 3PGA to cY-glycerophosphate. Phosphoglycolate determination. Phosphoglycolate (O-60 nmol) in 0.9 ml of 0.05 M bicine buffer, pH 8.3, was dephosphorylated by incubation with 1 unit of Escherichia cola’alkaline phosphatase at 37°C for 1 h and the reaction was terminated by immersion in a boiling-water bath for 3 min. After chilling in ice, a-dianisidine and horseradish peroxidase were added to each tube (see Fig. 3) and the tubes incubated at 30°C for 5 min. The reaction was initiated by addition of 0.17 unit of spinach glycolate oxidase in a final volume of 1.0 ml. The absorbance was read at 436 nm after 20 min against a reagent blank. RESULTS Determination of &BP in the Presence of 3-PGA and P-Glycolate by Measurement of NaOH-Labile Phosphate
Commercial RuBP is generally available at a purity of 70 to 85% but deteriorates on
AND SALUJA
RuBP
3-PGA , 0
of Phasphogiycoiote
1. 20
40 Time
0
SO
(Min)
FIG. 1. Time course of release of Pi from RuBP, 3PGA, and P-glycolate by 1 N NaOH at 25°C. RuBP (200 nmol) was incubated in 2.0 ml of 1.0 N NaOH. At specified times 0.2-ml samples were removed and neutralized with 0.1 ml of 2.0 N HsSO+ The concentration of 3-PGA and P-glycolate was 0.150 mM and both were treated as was RuBP. Alkali-labile Pi was determined as described in Table 1. The Pi present in unhydrolyzed samples has been subtracted.
storage. A preparation of RuBP, claimed to be 87% pure when assayed spectrophotometrically on the basis of ?I-PGA produced by RuBP carboxylase (9,1 S), was tested for the release of orthophosphate (Pi) by 1 N NaOH at 25°C in 30 min. Under these conditions, the release of Pi from RuBP was less than one per molecule. Under the same conditions of alkaline hydrolysis, Pi was not liberated from 3-PGA and P-glycolate. Moreover, the presence of 3-PGA and/or P-glycolate at OS- to 5-fold molar ratios to RuBP did not alter the release of Pi from RuBP. Only one of the two phosphate groups of RuBP was released during alkaline hydrolysis (Fig. 1), and the value of less than one Pi released per molecule of RuBP was therefore indicative of the purity of the preparation. On this basis the preparation of RuBP used was 74% pure. The preparation contained 0.26 mol Pijmol RuBP. As shown in Fig. 1 the release of Pi from RuBP by 1.0 N NaOH was complete in 20 min. Moreover, under the same conditions no Pi was released from P-glycolate or 3PGA. The time dependence of alkali-labile Pi release from RuBP (Fig. 1) was done 4 days after an earlier analogous assay, and in the interim the RuBP content decreased
RIBULOSE
0
1 20
BISPHOSPHATE
t 40 Time
eo
OXYGENASE
1
(Min)
FIG. 2. Time course of release of Pi from RuBP, 3PGA, and P-glycolate by alkaline phosphatase at 37’C. The reaction mixture (1 .O ml) contained 50 mM bicine, pH 9.4, 10 mM Me, and 0.4 mM RuBP, P-glycolate, or 3-PGA in a final volume of 1.O ml. After transferring 0.05 ml to an equal volume of 20% trichloroacetic acid (TCA), the reaction was initiated with 1 unit of calf intestinal alkaline phosphatase (1000 units/mg proteins) contained in 1 ~1 of water. At times indicated, 0.05-ml samples were transferred analogously to TCA. Aliquots of the WA-quenched samples were used for the Pi determination as described in the footnotes to Table 1. Due to the negligible quantity of protein added, no clarification of the samples was necessary.
from 80 to 74% and the Pi present increased from 0.20 to 0.26 mol/mol RuBP even though the preparation had been kept frozen at -20°C. From these results it is apparent that RuBP can be determined in the presence of both P-glycolate and 3-PGA. The liberation of only one phosphate group by alkali is in agreement with Paech et al. (16) but conflicts with the report of Horecker et al. (17) that both phosphate groups of RuBP are labile to alkali. The value of 2.8 min for the half-life of RuBP in 1 N NaOH reported by Horecker et al. (17) is close to the value obtained for the release of Pi from RuBP (Fig. 1) under identical conditions. Although P-glycolate and 3-PGA are refractory to alkaline hydrolysis at 25°C by 1 N NaOH, these compounds are quantitatively hydrolyzed by alkaline phosphatase (Fig. 2). Alkaline phosphatase released almost one equivalent of Pi per mole of P-glycolate or 3-PGA and accounted for the approximate 95% purity of these compounds. Both phosphate groups of RuBP were re-
161
STOICHIOMETRY
leased by alkaline phosphatase. The purity of a RuBP sample based on the hydrolysis of one of the two phosphate groups by alkali was found to be in perfect agreement with the release of two phosphate groups by alkaline phosphatase. In experiments where the hydrolyses with alkaline phosphatase were done with mixtures of RuBP, P-glycolate, and 3-PGA individually or in various combinations, the total Z’i liberated was equal to the sum of Pi liberated by individual components. Either calf intestinal or E. coli alkaline phosphatase could be used for the hydrolysis of RuBP, P-glycolate, or 3-PGA. The calf intestinal alkaline phosphatase was optimally active in the pH range of 9.4 to 10.4 in the presence of 1 mM Mg”, whereas the enzyme from E. coli yielded identical results between pH 8 and 8.6 and 1 and 20 mM Mg 2+. Similar results were also obtained with wheat germ acid phosphatase at pH 4.5 in the presence of 10 mM Mg’+. ~i~~~lties in the determination glycolate by Chemical Assay
of P-
The procedure described by Calkins (18) for the determination of glycolate by reaction with 2,7-dihydroxynapthalene in 33 N H2S04 in a boiling-water bath yielded about 6% of absorbance at 530 nm with P-glycolate as compared to an equivalent amount of glycolate. By conducting the above reaction at 121 “C in an autoclave for 15 min, a linear increase in the absorbance up to 200 nmol of P-glycolate was achieved. At this temperature, however, severe interference was caused by RuBP, Tris, Ammediol, EDTA, EGTA, or GSH, and slight interference was caused by 3-PGA. EDTA, EGTA, and Ammediol increased the intensity of absorbance without shifting the absorbance maxima, but the presence of RuBP completely masked the 535nm absorbance maxima and shifted them to 420 nm. Attempts to remove RuBP from a mixture containing RuBP, P-glycolate, and 3-PGA by reacting with dihydrox-
162
PUROHIT,
MC FADDEN.
yboryl-substituted cellulose ( 19) or by reacting with hydrazine-substituted resins were only partially successful or resulted in high dilution of P-glycolate, lowering its concentration almost beyond the sensitivity of the Calkins procedure. All attempts to eliminate the interference by the ribulose moiety using alkaline phosphatase-dephosphorylated synthetic reaction mixtures followed by the Calkins reaction at 100 or 121 “C yielded unsatisfactory results. Interference by the buffers could be eliminated by substituting bicine, Hepes, or TES buffers for Tris or Ammediol, as these buffers were unreactive at a concentration of 0.1 M in the sample assayed. The procedure of anion-change chromatography (1) was found to be of limited applicability for separation and routine assay of P-glycolate. Attempted Enzymatic Glycolate
Assays of P-
The assay of P-glycolate by dephosphorylation with alkaline phosphatase followed by rephosphorylation in the presence of pyruvate kinase, (Mg*+, K+) ATP, and phosphoenolpyruvate followed by estimation of the resulting pyruvate by lactate dehydrogenase and NADH seemed feasible. Assays were therefore attempted at several pH values from 7 to 8.3. Although glycolate-dependent oxidation of NADH was observed in the presence of 0.5 mM glycolate, the rate was too low to measure at 50 PM glycolate. Attempts to phosphorylate ADP with the donor P-glycolate in the presence of an excess of pyruvate kinase with parallel estimation of ATP using glucose, hexokinase, and glucosed-phosphate dehydrogenase was feasible at high P-glycolate concentrations and pH 8.3, but the reaction rate at 50 pM P-glycolate was too slow to be of use. Taking advantage of catalysis by lactate dehydrogenase of glycolate oxidation, we tried to pull the reaction toward glyoxylate in the presence of semicarbazide at pH 6.5 to 8.5. Although nonenzymatic glyoxylate
AND SALUJA
semicarbazone formation at pH 6 with known quantities of glyoxylate was quantitative, the coupled reaction rate with 50 pM glycolate was extremely slow. Attempted Determination of Glycolate Resulting from Dephosphorylation of PGlycolate as Glyoxylate Phenylhydrazone The oxidation of glycolate by glycolate oxidase results in the formation of H202 and glyoxylate (20), which may be further oxidized to CO*, H20, and formate by H202. All attempts to trap glyoxylate produced as the glyoxylate phenylhydrazone by the method of Baker and Tolbert (2 1) gave variable recoveries of glycolate derived from Pglycolate, even in the presence of 150 to 4000 units of catalase. However, the production of glyoxylate phenylhydrazone from authentic glyoxylate was quantitative as measured by the increase in absorbance at 324 nm. This reaction with glyoxylate was not influenced by the presence of alkaline phosphatase, catalase, and glycolate oxidase individually or in combination. In these experiments glycolate was generated by complete dephosphorylation of the tricyclohexylamine salt of phosphoglycolate by alkaline phosphatase. It is possible that cyclohexylamine might have been the cause of our unsatisfactory results, but this aspect was not investigated. Determination of Glycolate by Oxidation with Spinach Glycolate Oxidase and Measurement of the Resultant H202 The absorbance at 436 nm due to the chromophore resulting from the reaction of H202, generated stoichiometrically through catalysis by glycolate oxidase, with a-dianisidine in the presence of peroxidase obeyed Beer’s law for up to 60 nmol of authentic glycolate. Similar results were obtained for glycolate arising from complete hydrolysis of tricyclohexyl ammonium P-glycolate by alkaline phosphatase, but the absorbance
RIBULOSE
Glycolote
BISPHOSPHATE
OXYGENASE
(nmoles)
FIG. 3. Standard curve for glycolate. Sodium P-glycolate. was dephosphorylated in 1.0 ml of 0.05 M bicine (pH 8.3) by 1 unit of E. coli alkaline phosphatase for 1 h at 37°C. The tubes were heated in a boiling-water bath for 3 min, chilled in ice, and stirred briefly with a Vortex mixer. Aliquots of 0.9 ml containing the quantities specified were used for the determinations. To each tube, 0.085 ml of aqueous solution of 1.755 mM a-dianisidine 2 HCl* 2 Hz0 and 4.56 purpuroglin units of horseradish peroxidase in 0.015 ml of 0.1 M bicine buffer, pH 8.3, was added. After equilibration at 30°C for 5 min, the reaction was initiated by adding 5.0 ~1 containing 0.17 units of spinach glycolate oxidase. The absorbance was read at 436 nm in a Cary 14 spectrophotometer, after incubation for 20 min at 3O’C with correction for a reagent blank. In some experiments 100-400 nmol of 3-PGA was also present in the original alkaline phosphatase-treated mixture.
was about 20% lower than that obtained with authentic glycolic acid. This disparity disappeared when P-glycolate was used as the Na’ salt prior to dephosphorylation with alkaline phosphatase. The development of color was complete within 20 min at 3O”C, and further incubation up to 40 min did not alter the absorbance readings in the presence of 0, 5, or 20 mM Mg2+. Dephosphorylation of P-glycolate is a prerequisite of any method for glycolate determination using glycolate oxidase. In Fig. 2 it is shown that both 3-PGA and RuBP are also dephosphorylated by alkaline phosphatase. Thus, the enzymatically dephosphorylated reaction mixture contains variable amounts of ribulose, glycerate, and glycolate. In the absence of any information on the reactivity of ribulose and glyceric acid with glycolate oxidase and horseradish peroxidase, the interference by glyceric acid and
STOICHIOMETRY
163
ribulose in the glycolate assay was investigated. The absorbance at 436 nm produced by glycolate arising from P-glycolate is shown in Fig. 3. The slope obtained was in perfect agreement with the molar absorption coefficient of 8.3 X lo3 M cm-’ for the chromophore generated by the reaction of H202 with a-dianisidine in the presence of peroxidase (22) and was unaffected by the presence of 100 to 400 nmol of 3-PGA in the original mixture. Unlike glycerate, ribulose generated by alkaline phosphatase from RuBP increased the intercept values of the plots obtained with varying glycolate in the glycolate assay system (Fig. 4). The increase in the intercept values, however, was proportional to the RuBP originally present (see inset, Fig. 4), and no alteration of the slopes was noticed. A linear least-squares regression analysis of the absorbance at 436 nm in the presence
FIG. 4. Interference of ribulose in the determination of glycolate. From 9.4 to 56.4 nmol P-glycolate alone (8) or in the presence of 85 (O), 425 (A), and 850 (8) nmol RuBP (corrected for purity by enzymatic assay or by alkaline hydrolysis) were dephosphoryiated for 1 h at 37°C by 1 unit of E. coli alkaline phosphatase in 0.9 ml of 0.05 M bicine buffer, pH 8.3, containing 5 mM Mg’+. After heat quenching, the entire reaction mixture was used for color development as described in Fig. 3. Each curve was generated by linear least-squares regression analysis. The inset shows a plot of the y intercept vs concentration of ribulose bisphosphate (RuBP).
164
PUROHIT,
MC FADDEN, TABLE
STOICHIOMETRY
FOR
AND SALUJA 1
RuBP OXYGENASE
Experiment number
Enzyme source
O2 consumed (nmol)
Phosphoglycolate produced” (nmol)
1 2 3
Spinach Spinach Barley
127 223 172
135 240 213
Phosphoglycolate: oxygen 1.06 1.08 1.24
Note. The reactions were interrupted after 6 to 8 min of incubation and an aliquot portion rapidly transferred to a boiling-water bath. Thus, the period of phosphoglycolate production slightly exceeded the period of oxygen consumption. ’ Phosphoglycolate was measured by correcting the OD4s6 for absorbance derived from RuBP (Fig. 4). Residual RuBP was determined from the amount of alkali-labile phosphate remaining. When residual RuBP was measured by using H’%ZO; and running the RuBP carboxylase reaction to completion, the phosphoglycolate:02 ratios for experiments 1 to 3 were 1.07, 1.10, and 1.22, respectively.
of 0, 85, 425, and 850 nmol of ribulose with glycolate varying from 9.4 to 56.4 nmol/ml of the assay mixture yielded a mean slope of 0.008 + 0.0003, the uncertainty being at the level of 1 standard deviation. This value agreed closely with that obtained in the presence or absence of glycerate (Fig. 3).
Stoichiometry Oxygenase
Experiment number and enzyme sourcea (1) (2) (3) (4) (5) (6) (7)
Spinach minus RuBP control Minus enzyme control Spinach Spinach Spinach Spinach Spinach
(8) R rubrum
(9) Minus enzyme
control
STOICHIOMETRY
O2 consumed (nmol) 0 0 74 80 167 230 80 94 0
2 FOR
Phosphoglycolate produced* (nmol) 0 0 84 82 160 240 76 96 0
by RuBP
In Table 1, data establish that the measured P-glycolate:02 ratio was close to 1. This ratio slightly exceeded 1 because of the methodology used to stop both reactions. Of
TABLE COMPLETE
for Catalysis
RuBP OXYGENASE 3-PGA pdUWd (nmol)
Residual RuBP (nmol)
0 0 135 160 310 596 186 219 0
0 777 712 1530 590 1150 1475 1411 1528
Theoretical residual RuBP’ (nmol)
Phosphoglycolate: oxygen -
800 693 1479 563 1184 1468 1143 1600
1.14 1.03 0.96 1.04 0.95 1.02 -
o Reactions were quenched by the addition of acid. Incubations were from 1.1 to 7.5 min with 50 to 300 pg spinach enzyme added except in Exp. 2. In Exp. 8, 200 wg of R. rrtbrum enzyme were added. In Exp. 1, RuBP was not added to an otherwise complete reaction mixture containing 50 ug of spinach enzyme. to correct for residual RuBP. b Calculated from ODus as described for Table 1 using the enzymatic assay (with H”C0;) c Initial RuBP in Exps. 2, 3, and 5 was 800 nmol and in Exps. 4 and 6-9 was 1600 nmol. ’ Stoichiometrics for catalysis by RuBP carboxylase and oxygenasc are RuBP+ RuBP-’
+ COa + HsO * + O1 -+ 3-PGA-’
2(3-PGA-‘) + 2 H+ + P-glycolate-’ + 2 H+
Theoretical residual RuBP was calculated by (i) averaging oxygen consumed with P-glycolate produced to yield RuBP disappearing via RuBP oxygenase (RuBP,,); (ii) subtracting RuBP.., from 3-PGA total to yield 3-PGA appearing via RuBP carboxylase (3-PGA&; (iii) halving 3-PG&.to yield RuBP disappearing via RuBP carboxylase (RttBP..,); and (iv) theoretical residual RuBP = RuBPr,,ru., - (RuBPee, + RuBP,,).
RIBULOSE
BISPHOSPHATE
OXYGENASE
particular interest is the fact that ODdj6 values can be corrected for contributions due to unreacted RuBP measured either enzymatically or chemically by release of Pi by alkali. The latter methods were in excellent agreement (Table 1). In Table 2, data establish that the measured P-glycolate:02 ratio was very close to 1 when the RuBP oxygenase reaction was terminated with acid. Measured values of residual RuBP were in excellent agreement with theoretical values for residual RuBP when a correction was made for RuBP utilization by the simultaneous competing reaction catalyzed by RuBP carboxylase (Table 2). Analysis of the data in Table 2 reveals that the mole ratio of RuBP disappearing via RuBP oxygenase to that disappearing via RuBP carboxylase is 1.4-3.1 under the experimental conditions employed. DISCUSSION
A frequently used assay for the oxygenase activity of RuBP carboxylase simply involves the measurement of RuBP-dependent O2 consumption. This method is not fully adequate, however, because reaction products are not measured. Although a good enzymatic analysis of one of the products, 3PGA, exists, this compound is also produced by RuBP carboxylase. Unfortunately, prior to the present work there was not a good assay available for the second, and unique product, P-glycolate. We have described here such an assay; in addition to enabling facile measurement of the stoichiometry for the RuBP oxygenase reaction, this assay yields results in excellent agreement with those of Lorimer and colleagues (2,3). After termination of oxygen consumption, residual RuBP, P-glycolate, and 3-PGA can be measured in separate aliquot portions of a single reaction mixture. The results not only specifically measure the stoichiometry for catalysis by RuBP oxygenase but also allow a crude estimate of RuBP oxygenase:
STOICHIOMETRY
165
carboxylase ratio under the conditions employed. For example, these ratios varied between 1.4 and 3.1 in the presently described research. It is crucial to note, however, that bicarbonate was used at a sufficient concentration to activate fully both RuBP oxygenase and carboxylase, yet was diluted 19- to 30-fold to a final concentration of 6 pM CO2 to minimize inhibition of the former activity by COZ. Thus, under these conditions the concentration of CO* severely limited the rate of the RuBP carboxylase reaction. Clearly, it is impossible to obtain near maximal velocities for both oxygenase and carboxylase when measured simultaneously (23,24).
The ratio of RuBP oxygenase:carboxylase for the spinach enzyme is normally around 0.13 when the oxygenase is measured at 250 PM 0, under otherwise optimal conditions (25,26). The present methodology could be easily modified to measure RuBP carboxylase under optimal conditions. For example, after the enzymatic assay of 3-PGA, the addition of saturating HCO; should yield a rate of NADH oxidation that is maximal and proportional to RuBP carboxylase, providing residual RuBP is saturating and all other components in the coupled enzyme assay mixture are in excess. It is shown that RuBP can be determined quantitatively using RuBP carboxylase and measuring the incorporation of H14CO; incorporation into 3-PGA. Results are in excellent agreement with those yielded by decomposition of RuBP by alkali. Both assays are specific and other components in the reaction mixture containing RuBP carboxylase/oxygenase do not interfere. An elegant alternative for measuring the stoichiometry for the RuBP oxygenase reaction has recently been described (23). This method is based upon use of [ 1-3H]RuBP and measurement of oxygen uptake and of [ 3H]P-glycolate production after separation by anion-exchange chromatography. Use of H14CO; to activate the oxygenase also enables the determination of [ 14C]P-glycerate,
166
PUROHIT,
MC FADDEN,
which is a measure of the competing carboxylase reaction. Initial and residual RuBP are also measured, as is total 3-PGA produced. The resultant stoichiometry is good, but the method is cumbersome in that [l3H]Ru13P must be prepared and P-glycolate must be isolated. The methods described enable reliable assays for RuBP oxygenase. Although each study of oxygen ~nsumption requires an oxygen electrode, durations are short and up to 20 rates/h can be measured. Many subsequent assays of reactant stoichiometry can be simuitaneously performed. Thus, the methodology enables a large number of assays per day. We recommend that iaboratories use this technique to randomly check the reliability of faster assays in which RuBP-de~ndent oxygen consumption is measured. ACKNOWLEDGMENT Much of this man~c~pt was completed at the Technical University of Munich (TUM), Freising-Weihenstephan, where B.A.M. was a Humboldt Senior Scientist. He thanks Berthold Hock for the many courtesies extended while a guest investigator at the TUM.
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