0 10 20 0 Retention time (min)

20 0

Figure 9.79 Chromatograms of («4) standard succinyl-CoA, obtained by injecting 0.6 nmol of standard solution (retention time for succinyl-CoA peak, 16.3 min), (B) blank, and (C) sample (20 /¿L of brain mitochondria containing 0.068 mg of protein). Arrow in the blank points to the retention time of succinyl-CoA. (From Shylaja et al., 1990.)

9.7.12 Sucrose Phosphate Synthetase (Salvuccl and Crafts-Brandner, 1991)

Sucrose phosphate synthetase catalyzes the reaction of UDP-glucose with fructose-6-P to form sucrose-6-P and UDP. This step is the penultimate step in the synthesis of sucrose in leaves. The chromatographic method can be applied to many UDP-glucose-requiring enzymes. The method eliminates the need for treatment of reaction mixtures with alkaline phosphatase.

Radiolabeled UDP-glucose and sucrose-P were separated on a Selectispher-10 boronate column (5 mm x 250 mm). The mobile phase was 0.12 M sodium phosphate (pH 7.6) delivered at a rate of 1 mL/min. The column eluent was monitored for absorbance at 262 nm, and for radioactivity by a radioactive flow-through detector.

Sucrose phosphate synthetase was assayed at 30°C in a total of 50 fiL containing 50 mM Hepes-KOH (pH 7.5), 15 mM MgCl2,1 mM EDTA, 6 mM UDP-[14C]glucose (1 mCi/mmol), 3 mM fructose-6-P, 15 mM glucose-6-P, and enzyme. Immediately prior to termination by boiling, 90 ¡jlL of 50 mM EDTA was added to prevent metal-dependent hydrolysis of UDP-glucose. After 3 minutes at 100°C, the quenched assay was cooled and treated with 0.1 mL of Dowex AG-50W to remove components that might decrease the life of the HPLC column. The mixture was centrifuged, and an aliquot of the supernate was taken for analysis by HPLC. Salvucci and Crafts-Brandner also described assays for sucrose synthetase and UDP-glucose pyrophosphorylase.

Sources of enzyme were tobacco leaf tissue or red beet tubers homogenized at 4°C in 50 mM Hepes-KOH (pH 7.2), 5 mM MgCl2, 1 mM EDTA, 25 mM mercaptoethanol, 1% (w/v)polyvinylpyrollidone-40,1 mM phenylmethylsulfo-nyl fluoride, and 10 (¿M leupeptin. Supernates obtained by centrifugation were desalted and equilibrated with a buffer containing all but the last three components in the homogenization medium.

9.7.13 6-Phosphogluconate Dehydratase (Taha and Deits, 1994)

6-Phosphogluconate dehydratase participates in the Entner-Doudoroff pathway, which plays a primary role in glucose metabolism in many microorganisms. The enzyme catalyzes the dehydration of 6-phosphogluconate to form 2-keto-3-deoxy-6-phosphogluconate.

The product, 2-keto-3-deoxy-6-phosphogluconate, was separated by chromatography at room temperature and a flow rate of 1 mL/min on a Dionex CarboPac PA-1 column (4 mm x 250 mm). The mobile phase was composed of 24 mM NaOH and 300 mM sodium acetate for 5 minutes, followed by a linear gradient to 700 mM sodium acetate in 10 minutes. A linear gradient back to the initial conditions was run in 5 minutes. Pulsed amperometric detection was used with a pulse train consisting of a 480 ms detection pulse at +80 mV, followed by pulses of 120 ms at +600 mV and 60 ms at -600 mV.

The reaction mixture was composed of 10 mM Bicine (pH 8.0) containing 50 mM NaCl, 10 mM /3-mercaptoethanol, 2 mM MnCl2, and 1.2 mM 6-


Figure 9.80 Representative chromatographs of Entner-Doudoroff metabolites, (a) 6-Phosphogluconate dehydratase-catalyzed formation of 2-keto-3-deoxy-6-phosphogluconate (12.51 min). (b) Blank run for 6-phosphogluconate dehydratase-catalyzed formation of 2-keto-3-deoxy-6-phosphogluconate, (KDPG); 6-phosphogluconate omitted from assay. (From Taha and Deits, 1994.)

phosphogluconate. Samples were quenched by the addition of trichloroacetic acid to give a final concentration of 5%. After centrifugation, a volume equivalent to 9% of the sample volume, containing 4 M NaOH and 2 M sodium acetate, was added to neutralize the trichloroacetic acid and lower the pH to about 5. A 20 fiL sample was used for HPLC analysis. In contrast to a spectrophotometric coupled-enzyme assay, which showed an initial lag phase, the HPLC-based assay was linear for up to 2 minutes. Product formation was also linear with the amount of enzyme added.

Enzyme used in the assays was purified from Azotobacter vinelandii. A modification of the assay was used to follow the approach to equilibrium of the 2-keto-3-deoxy-6-phosphogluconate aldolase reaction.

Figure 9.80 shows representive chromatograms.


9.8.1 A5-3/3-Hydroxysterold Dehydrogenase (Suzuki et al., 1980)

The activity A5-3/3-hydroxysteroid dehydrogenase catalyzes the conversion of pregnenolone to progesterone, the progestational hormone of the placenta and corpus luteum. The product has an absorption maximum at 240 nm, and therefore detection can be readily carried out by UV absorbances near this wavelength.

In this assay the amount of product formed during the reaction was determined on a reversed-phase HPLC (/iBondapak) column containing cyanopro-



Retention time (min)

Figure 9.81 HPLC chromatograms of steroids. The elution profiles of five standard A4-3-oxosteroids are illustrated as the broken line. Peaks = A, progesterone; B, 17 a-hydroxyprogesterone; C, androstenedione; D, testosterone; E 11-deoxycortisol. The solid line is a chromatogram of a defatted extract obtained by incubation of pregnenolone with ovarian homogenates. (From Suzuki et a]., 1980.)

pylsilane as the functional group. The column was eluted isocratically with a mixture of acetonitrile and water (1:4, v/v). Detection was at 254 nm.

The substrate, pregnenolone (158 nmol) dissolved in propylene glycol, was added to the incubation flask containing the enzyme preparation and NAD in a final volume of 5 mL. After an incubation period of 60 minutes, the reaction was terminated by addition of 15 ¡jlL of dichloromethane, and radioactive progesterone was added as a recovery standard. The organic phase was recovered, dried, and redissolved in 70% ethanol, and a sample was analyzed by HPLC. Figure 9.81 shows an analysis of an incubation mixture with and without incubation with the ovarian preparation. The substrate (not seen) is converted exclusively to progesterone.

The ovaries of rats were used in the preparation of the active fraction.

9.8.2 11-0-Hydroxylase and 18-Hydroxylase (Gallant et al., 1978)

The ll-/3-hydroxylase found in the adrenal cortex catalyzes the hydroxylation of 11-deoxycorticosterone to corticosterone. The enzyme requires NAD as a cofactor and contains heme as the prosthetic group.

The substrate, 11-deoxycorticosterone, was separated from the two reaction products, corticosterone (ll-/3-hydroxylation) and 18-hydroxyl-ll-deoxycorticosterone (18-hydroxylation), on reversed-phase HPLC (MicroPak silica) with a mobile phase of 16% tetrahydrofuran in water. Figure 9.82/1 shows a chromatogram of the separation of the authentic steroids.

Hydroxylase activity was determined in a reaction mixture containing mitochondrial protein and 11-deoxycorticosterone (60 fiM). The reaction was started by the addition of 10 mM isocitrate as the source of reducing equivalents. At intervals during the incubation, samples were removed

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