[22] Measurement of the intermediates of the photosynthetic carbon reduction cycle, using enzymatic methods

[22]

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261

[22] Measurement of the Intermediates of the Photosynthetic Carbon Reduction Cycle, Using Enzymatic Methods By ERWIN LATZKO and MARTIN GIBBS Preparation of the Extract For intact chloroplasts 1 and unicellular a l g a e / r a p i d mixing with 20% HC104 at 0 ° and additional full power sonic oscillation for 30 seconds is recommended. After removal of the precipitate by centrifugation for 5 minutes at 7000 g, the supernatant fluid is carefully titrated potentiometrically with 5 M K~CO~ to pH 5.5. The precipitated KClO4 is removed by centrifugation for 3 minutes at 3000 g. In the clear extract, glyceraldehyde S-P, dihydroxyacetone-P, ATP, ADP, and AMP should be measured within 2 hours; ribose 5-P, ribulose 5-P, xylulose 5-P, ribulose 1,5-P2, glucose l-P, glucose 6-P, fructose 6-P, fructose 1,6-P2, sedoheptulose 1,7-P2, glycerate S-P, and glycolate are stable for at least 2-3 days if the extract is stored a t - 2 0 °. Whole leaves are immersed in liquid nitrogen and pulverized with a mortar. 1"3 Thawing has to be avoided until the powder is suspended in 5% HC104. The brei is sonicared for 30 seconds and further treated as described for chloroplasts.

Reagents ]. H C I O 4 , 20% w/v II. K2COa, 5 M

D-Ribulose 1,5-P2, D-Ribulose 5-P, D-Ribose 5-P, and n-Xylulose 5-P Assay Principle. 4 Since the concentration of ribulose 1,5-P2 is extremely low in most plant preparations, 1 a quantitative enzymatic-optical assay is not feasible. We have used an isotopic assay involving the incorporation

IE. Latzko and M. Gibbs, Plant Physiol. 44, 396 (1969). 2E. Latzko and M. Gibbs, in "Methoden der enzymatischen Analyse" (H. U. Bergmeyer, ed.), 2nd ed. Verlag Chemie, Weinheim, 1970. , 1970. 3U. Heber, K. A. Santarius, M. A. Hudson, and U. W. Hallier, Z. Naturforsch. B 22, 1189 (1967). 4E. Latzko and M. Gibbs, in "Methoden der enzymatischen Analyse" (H. U. Bergmeyer, ed.), 2nd ed. Verlag Chemie, Weinheim, 1970. 1970.

262

METHODOLOGY

[22]

of 14CO2 by ribulose 1,5@2 carboxylase (Eq. 1). The pentose monophosphates are converted into ribulose 1,5-P2 by purified enzyme-reactions (Eqs. 2-4) and measured via Eq. 1. With a standard curve, the lower limit of the determination is < 5 nmoles of pentose phosphates per assay. Ribulose 1,5-P~ + 14CO2 + H 2 0 Ribulose 5-P + A T P Ribose 5-P

ribulose 1,5-P2) 2 glycerate 3-PJ4C carboxylase

ribulose 5-P) ribulose 1,5-P2 + ADP kinase

ribose5-P~ ribulose 5*P isomerase

Xylulose 5-P 3-epimerase xylulose5-P~ribulose 5-P

(1) (2) (3)

(4)

Reagents III. Triethanolamine-HCl buffer, 0.4 M, in 0.2 M EDTA, pH 7.6 (tra-EDTA buffer) IV. MgCI2, 0.5 M V. NaHliCO3, 0.5 M (5 mCi 14C/mmole of COs) VI. HCI, 1.0 M VII. KC104 solution saturated at 0 ° VIII. Reduced glutathione, 0.1 M IX. ATP, 0.05 M (~- pH 5.5) X. Ribulose 1,5-P2, 0.001 M XI. Ribose 5-P, 0.01 M XII. Ribulose-l,5-P2 carboxylase 5 ( - 20 units/ml) free of phosphoenolpyruvate carboxylase, transketolase, and transaldolase XIII. Ribulose-5-P kinase 6 (= 40 units/ml) XIV. Ribose-5-P isomerase 7 (= 50 units/ml) XV. Xylulose-5-phosphate 3-epimerase 8 ( 2 50 units/ml) (XIIIXV free of transketolase and transaldolase; prepared according to the references; diluted from stock solutions with 0.05 M Tris buffer, pH 7.6, before use) XVI. Tris buffer, 0.05 M, pH 7.6 Procedure for Ribulose 1,5-Pz. The following solutions are mixed in the described order in a clinical centrifuge tube: A. 0.25 ml of extract, 0.02 ml of HCI* (* = after addition of this solution the assay tubes have to be carefully and vigorously ~E. Racker, Vol. V, p. 266. nJ. Hurwitz, Vol. V, p. 258.

7E. Racker, Vol. V, p. 280. 8M. Tabachnick, P. A. Stere, J. Cooper, and E. Racker, Arch. Biochem. Biophys. 74, 315 (m58).

[22]

P H O T O S Y N T H E T I C C A R B O N R E D U C T I O N CYCLE

263

shaken), 0.075 ml of tra-EDTA buffer*, 0.01 ml of MgCl~, 0.1 ml of H20, 0.02 ml of ribulose-l,5-P2 carboxylase*, 0.02 ml of NaH14CO3. B. In the blank control the extract is replaced by 0.25 ml of solution VII. C. The standard curve is prepared using 0.001-0.05 /.~mole of ribulose 1,5-P2 per assay. The assay mixture is incubated for 30 minutes at 20 °. The reaction is stopped by the addition of 0.1 ml of 20% HC104. The precipitate is centrifuged off. An aliquot of the supernatant fluid is distributed homogeneously on a planchet, which is covered with one layer of lens tissue, dried under an infrared lamp, and assayed for radioactivity. Procedure for o-Ribose 5-P, o-Ribulose 5-P, and n-Xylulose 5-P. Since the content of pentose monophosphates has to be corrected for ribulose 1,5-Pz, both assays, ribulose 1,5-P2 and pentose monophosphates, are carried out simultaneously and with the same solutions. Analogous to the ribulose 1,5-P2 assay, the following solutions are mixed in the described order: D. 0.25 ml of extract, 0.02 ml of HCI* (* after addition of this solution the assay tubes have to be shaken), 0.075 ml of tra-EDTA buffer*, 0.01 ml of MgC12, 0.02 ml of ribulose-l,5-P2 carboxylase*, 0.02 ml of NaH14CO3*, 0.01 ml of ATP, 0.02 ml of reduced glutathione, 0.02 ml of ribulose-5-P kinase*, 0.02 ml of ribose-5-P isomerase*, 0.02 ml of xylulose-5-P epimerase*. E. In the blank control the extract is replaced by 0.25 ml of solution VII. F. The standard curve is measured using 0.01-0.1 p.mole of ribose 5-P per assay. Further treatments as described for ribulose

1,5-P2. Definition of Unit and Calculations. One unit of enzyme converts 1/~mole of substrate per minute. Calculations for ribulose 1,5-P2 content per 0.25 ml extract: cpm for A - (cpm for B) -----cpm for ribulose 1,5-Pz. Calculations for pentose monophosphates per 0.25 ml extract: cpm for D (cpm for E + cpm for the ribulose 1,5-P2 content) = cpm for pentose monophosphates.

264

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D-Glyceraldehyde-3-P, Dihydroxyacetone-P, D-Fructose- 1,6-P2 and D-Sedoheptulose- 1,7-P2

Assay Principle? The combined enzymatic-optical assay is based on the dehydrogenation of NADH by the glycerol-l-P dehydrogenase reaction (Eq. 5). Glyceraldehyde 3-P, fructose 1,6@2 + sedoheptulose 1,7-P2 are transferred into dihydroxyacetone-P in sequential steps (Eqs. 6-8). Dihydroxyacetone-P + NADH Glyceraldehyde 3-P Fructose 1,6-P2

aldolase

Sedoheptulose 1,7-P2

glycerol-l-P) glycerol I-P + NAD dehydrogenase

triose-P ) dihydroxyacetone-P isomerase

(5) (6)

glyceraldehyde 3-P + dihydroxyacetone-P

(7)

aldolase ) dihydroxyacetone-P+ erythrose 4-P

(8)

)

Reagents XVII. NADH, 0.005 M XVIII. Glycerol-l-P dehydrogenase (= 50 units/ml) XIX. Triose-P isomerase (2 50 units/ml) XX. Fructose-l,6-Pz aldolase (= 50 units/ml) (XVIII-XX commercial preparations are diluted with 0.05 M Tris, pH 7.6 before use.)

Procedure. In a final volume of 1 ml, the following solutions are mixed in a microquartz cell with a 10-mm light path: 0.05 ml of tra-EDTA buffer (solution IlI), extract, 0.02 ml of NADH, 0.02 ml of glycerol-l-P dehydrogenase (AAa40 = dihydroxyacetone-P); 0.02 ml of triose-P isomerase, (AA340 = glyceraldehyde 3-P); 0.02 ml fructose-l,6-P2 aldolase (AAa40 = dihydroxyacetone-P + glyceraldehyde 3-P from fructose 1,6-P2 and dihydroxyacetone-P from sedoheptulose 1,7-P2). The reference cuvette contains instead of the extract a suitable volume of solution VII. The AA at 340 nm of the reference cuvette is subtracted from the AA of the assay cuvette. Calculations. (AA340 nm)/6.2 = ~mole per volume of extract used for the assay. ~T. Bucher and H. J. Hohorst, in "Methoden der enzymatischen Analyse" (H. U. Bergmeyer, ed.), 1st ed., p. 246. Verlag Chemie, Weinheim, 1962.

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REDUCTION

CYCLE

265

D-Glucose 6-P, D-Glucose l-P, D-Fructose 6-P, and D-Fructose 1,6-P2 or A T P

Assay Principle) T M T h e combined enzymatic-optical assay is based on the reduction o f NADP by the glucose-6-P d e h y d r o g e n a s e reaction (Eq. 9). Glucose l-P, fructose 6-P, and fructose 1,6-P2 or A T P (Eqs. 10-13) are assayed with glucose 6-P in sequential steps. Glucose 6-P + NADP

glucose-6-P dehydrogenase

Glucose 1-P phosphogl. . . . .

6-P-gluconate + NADPH

Fructose 1,6-P2 ATP + glucose

glucose 6-P

(10)

~ glucose 6-P

(1 l)

tase

Fructose 6-P hexose-Pis. . . . . . .

e

C-l-fructose ~ fructose 6-P + Pi

diphosphatase hexokmase

(9)

; ADP + glucose 6-P

(12) (13)

C-l-Fructose diphosphatase isolated from spinach leaves ~2 does not react with sedoheptulose 1,7-P~. T h e K m for fructose 1,6-P2 is about 0.3 m M which is lower than the Km o f rabbit muscle aldolase for fructose 1,6-P~ (= 0.75 mM). For extremely low concentrations o f fructose 1,6-P2, assay by means o f Eq. (12) r a t h e r than Eq. (7) is r e c o m m e n d e d . Reagents XXI. XXII. XXIII. XXIV. XXV. XXVI. XXVII. XXVIII. XXIX.

Tris.HC1 buffer, 1.0 M, p H 8.5 NADP, 0.005 M Glucose, 0.1 M EDTA, 0.1 M, p H 8.5 Glucose-6-P d e h y d r o g e n a s e (= 50 units/ml) Phosphoglucomutase (= 50 units/ml) Hexose-6-P isomerase (= 50 units/ml) Hexokinase (= 50 units/ml) C-l-fructose diphosphatase TM (50 units/ml) free o f transaldolase and aldolase XXX. T r i s ' H C l buffer, 1.0 M, p H 7.6 ( X X V - X X I X are diluted before.' use with 0.05 M T r i s . H C l buffer, p H 7.6 X X V - X X V I I ! are commercial preparations.) I°H. J. Hohorst, in "Methoden der enzymatischen Analyse" (H. U. Bergmeyer, ed.), 1st ed., p. 134. Verlag Chemie, Weinheim, 1962. HE. Racker, in "Methoden der enzymatischen Analyse" (H. U. Bergmeyer, ed.), 1st ed., p. 160. Verlag Chemie, Weinheim, 1962. I~E.Racker, Vol. V, p. 272.

266

M~THO~)OLOGV

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Procedure for Hexose Monophosphates and Fructose 1,6-P2. In a final volume of 1.0 ml, the following solutions are mixed in a microquartz cell with 10-mm light path: 0.1 ml of Tris-buffer (sol. XXI), extract, 0.05 ml of NADP, 0.02 ml of MgCi2 (sol. IV), 0.02 ml of glucose-6-P dehydrogenase (&4340= glucose 6-P), 0.02 ml of hexose-6-P isomerase (AA340= fructose 6-P), 0.02 ml of P-glucomutase (AA340 = glucose l-P), 0.02 ml of EDTA, 0.02 ml of C-l-fructose diphosphatase (AA340= fructose 1,6-P2). In the blank cuvette the extract is replaced by an equal volume of solution VII. Procedure for ATP. ATP may be determined instead of fructose 1,6-P2 if EDTA and C-l-fructose diphosphatase are omitted from the abovementioned procedure and 0.05 ml of glucose and 0.02 ml of hexokinase are added instead ( ~ 3 4 0 = ATP). If solely ATP is assayed, the following solutions are added per milliliter: 0.1 ml of Tris-buffer (sol. XXX), extract, 0.05 ml of NADP, 0.02 ml of MgCI2 (sol. IV), 0.02 ml of hexokinase (~x~340 = ATP). In the blank cuvette the extract is replaced by an equal volume of solution VII.

D-Glycerate 3-P Assay Principle. 13 Two glycolytic back reactions (Eqs. 14 and 15) are coupled to reduce glycerate 3-P to glyceraldehyde 3-P consuming stoichiometric amounts of ATP and NADH. Glycerate 3-P + ATP

3-P-g]ycerate kinase ' glycerate 1,3-P2 + ADP

(14)

glyceraldehyde3-P Glycerate 1,3-P2 + NADH dehydrogenase ~ glyceraldehyde 3-P+ NAD ( 1 5 )

Reagents XXXI. Glycerate 3-P-kinase (= 50 units/ml) XXXII. Glyceraldehyde 3-P-deb.ydrogenase (2 50 units/ml) These enzymes (XXXI, XXXII) are diluted from commercial preparations with 0.05 M Tris-buffer, pH 7.6. In a final volume of 1.0 ml, the following solutions are added into a microquartz cell with a 10-mm light path: 0.1 ml of tra-EDTA buffer (sol. III), extract, 0.02 ml of NADH (sol. XVII), 0.1 ml of ATP (sol. IX), 0.02 ml of MgC12 (sol. IV), 0.02 ml of glyceraldehyde 3-P dehydrogenase, 0.02 ml of glycerate 3-P kinase. In the blank cuvette the extract is replaced by solution VII. 13T. Bucher, Vol. I, p. 415.

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267

Adenosine Diphosphate and Adenosine Monophosphate Assay Principle.TM The determination of ADP and AMP is coupled with NADH oxidation by the lactate dehydrogenase reaction (Eq. 17). In this series of reactions (Eqs. 16-18), 1 mole of oxidized NADH equals 1 mole of ADP or 0.5 mole of AMP. ADP + P-enolpyruvate pyruvate pyruvate + ATP kinase Pyruvate + NADH lactatedehydrogenas~ lactate + NAD AMP + ATP

myokinase~ 2 ADP

(16) (17) (18)

Reagents XXXIII. P-enolpyruvate, 0.05 M, pH 5.5 XXXIV. ATP, 0.05 M (pH 5.5, free of ADP -4- UDP -4- AMP) XXXV. KC1, 1.0 M XXXVI. Lactate dehydrogenase ( - 100 units/ml) XXXVII. Pyruvate kinase (= 50 units/ml) XXXVIII. Myokinase (= 50 units/ml) (XXXVI-XXXVIII are commercial preparations and are diluted with 0.05 M Tris-buffer, pH 7.6.)

Procedure. The following solutions are mixed in a microquartz cell to a final volume of 1.0 ml: 0.1 ml of tra-EDTA buffer (sol. III), extract, 0.02 ml of NADH (sol. XVII), 0.02 ml of lactate dehydrogenase (fie/340= pyruvate), 0.02 ml of P-enolpyruvate, 0.05 ml of KC1, 0.02 ml of MgClz (sol. IV), 0.02 ml of pyruvate kinase (2tA340 = ADP), 0.01 ml of ATP (sol. XXXIV), 0.02 ml of myokinase (2w/340/2= AMP). In the reference cuvette the extract is replaced by solution VII. Glycolate Assay Principle. The assay depends on the reduction of 2,6-dichlorophenolindophenol (DPIP) as catalyzed by glycolate oxidase (Eq. 19). Since this enzyme can also react with glyoxylate, the sensitivity of the method is increased if the glycolate concentration exceeds 0.5 mM. Therefore a standard curve rather than a coefficient factor is required in this assay. CH2OH--COOH + DPIP glycolat%C H O - - C O O H + DPIPH~ oxidase

(19)

14H. Adam, in "Methoden der enzymatischen Analyse" (H. U. Bergmeyer, ed.), 1st ed., p. 573. Verlag Chemie, Weinheim, 1962.

268

METHODOLOGY

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Reagents XXXIX. XL. XLI. XLII. XLIII. XLIV.

Tris-HC1 buffer, 1.0 M, pH 8.0 DPIP, 0.01% KCN, 0.1 M, in 0.01 M NH4OH FMN, 0.05 M (flavin mononucleotide) Glycolate oxidase TM diluted with 0.01 M phosphate buffer, pH 8.0, to = 50 units/ml Sodium glycolate, 0.01 M

Procedure. In a final volume of 1.0 ml, the following solutions are placed in a microquartz cell with a 10-mm light path: 0.1 ml Tris-buffer, extract, 0.1 ml of DPIP, 0.01 ml FMN, 0.003 ml KCN, 0.04 ml glycolate oxidase. The blank cuvette contains solution VII instead of the extract. The reaction is followed at 587 nm, T = 20 °. The standard curve is prepared using 0.025-0.25 Izmole of glycolate. Since glycolate oxidase can react with glycolate (Kin = 0.38 mM) or with glyoxylate (Kin = 2.5 raM), the standard curve is not linear below 0.15 izmole of glycolate per milliliter. Ascorbate or other reducing compounds which could react with DPIP must be omitted from the reaction or extraction medium. 15I. Zelitch, Vol. I, p. 528.

[23] Light Intensity Measurement; Characteristics of Light Sources; Filters and Monochromators By RICHARD

W . TREHARNE

Light Intensity M e a s u r e m e n t

Types of Light Measurement The term "light" strictly can be applied only to the visible portion of the electromagnetic spectrum. Since radiation outside of the visible range also is of importance for photoreaction studies, we shall broaden the concept of light measurements to include all measurements of radiant power in the ultraviolet, visible, and infrared portions of the electromagnetic spectrum. The words "light" and "radiant power" thus will be used interchangeably. There are three basic types of radiant power measurements and, as a further complication, each measurement can be expressed in two distinct types of units-radiometric or photometric units. The three types