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[43b] Assay of shikimic acid
352
AROMATIC AMINO ACIDS
[43a]
and finally with 500 ml of 5% unacidified ethanol. Shikimate-5-P was eluted with 2500 ml of 5% ethanol, 3 liters of 10% ethanol, and 2 liters of 25% ethanol. The fractions were combined and concentrated in a vacuum. The residual colorless syrup was diluted with water to 150 ml, and was found to contain 27 millimoles of shikimate-5-P. T h e solution was adjusted to pH 7 with NH4OH and treated with 100 ml of 0.5 M barium acetate. Absolute ethanol (950 ml) was added to give 80% (v/v) ethanol, and the solution was allowed to stand overnight at 0 °. T h e precipitate was collected by centrifugation, washed twice with 200 ml of 75% ethanol and once with 200 ml of absolute ethanol, and dried over P2Os under vacuum yielding 12.8 g (25 millimoles) of the barium salt as a white powder. Continued elution of the column with 10 liters of 25 % ethanol gave a slightly yellow syrup (assaying 7.9 g of shikimate5-P) from which was isolated 2.9 g of slightly tan barium salt. Both preparations of shikimate-5-P gave only one spot in descending chromatography on paper with butanol-I/acetic acid/water (100:6:2, v/v), 3 and with propanol-l/15 M N H 4 O H / H 2 0 (6:3:1, v/v). 4 However, a minor phosphorylated impurity appeared in paper electrophoresis for 1 hour at pH 5.5 and 900 volts (0.1 M sodium acetate buffer; length of paper 50 cm). An acid molybdate spray 4 was used to detect phosphate esters. Chromatography on Dowex-1 CI-X8 (200-400 mesh) at pH 9 (0.01 M Tris-Cl) with a lithium chloride gradient gave one symmetrical peak. The minor phosphate ester obtained in paper electrophoresis at pH 5.5 may have resulted from isomerization of the shikimate 5-phosphate to the 4-phosphate. 1 Hydrolysis of both barium salts with alkaline phosphatase gave rise to 1.0 mole of shikimate and 1.0 mole of Pl per equivalent weight of 500.1 sj. A. Fewster, Biochem.J. 85,388 (1962). ~;. S. Hanes and F. A. Isherwood, Nature 164, 1107 (1949).
[43b] Assay of Shikimic Acid By R. C. MILLICAN1 This assay2 depends on the treatment of shikimic acid with periodate 1Deceased-- March 7, 1964 2This procedure was described by R. C. Millican, Anal. Biochem. 6, 181 (1963). It is based on the procedure developed by L. Warren for the thiobarbituric acid assay of sialic acids [J. Biol. Chem. 234, 1971 (1959); Vol. VI [67]. Saslaw and Waravdekar (Biochim. Biophys. Acta 37, 367, 1960) have also published a method for shikimic acid determination, based on the thiobarbituric acid procedure.
[43b]
SHIKIMIC ACID ASSAY
353
in strong acid solution, followed by the reaction of the oxidation product with thiobarbituric acid. T h e chromogen is extracted into cyclohexanone to produce a stable color with a molar extinction of 33,000 at 535 m/~.
Reagents Shikimic Acid 4.28% Sodium metaperiodate in 9 M'H3PO4 10% Sodium arsenite in a solution of 0.5 M sodium sulfate-0.1 N sulfuric acid. 0.6% Thiobarbituric acid in 0.5 M sodium sulfate, adjusted to pH 7 with 5 N NaOH. Some material is insoluble, but this does not affect the color. Cyclohexanone
Procedure. To each sample containing up to 0.06 micromole of shikimic acid in a volume of 0.2 ml, 0.1 ml of periodate solution is added. The tubes are shaken and incubated at 80 ° for 5 minutes. At the end of the periodate oxidation 0.5 ml of arsenite solution is added and the tubes are shaken until the yellow-brown color disappears) Thiobarbituric acid solution, 2.5 ml, is added to each tube; the tubes are shaken, capped with a marble bead, and then heated in a vigorously boiling water bath for 15 minutes. During cooling the red color fades and the solution often becomes cloudy. This does not interfere with the final reading. One milliliter of this solution is transferred to another tube containing 1 m~ of cyclohexanone. The latter tube is ~ h a ~ n twice and centrifuged in a clinical centrifuge for 3 minutes. The clear upper cyclohexanone phase is red. The colors are very stable in the cyclohexanone phase. Optical densities are determined on the organic phase at 535 m g in a Beckman model,DU spectrophotometer with a 1-cm light path. Measurements are read against water. The color produced increases linearly between 0.01 and 0.06 micromole of added shikimic acid. The amount of shikimic acid present in a given sample can be determined from the following equation: micromoles shikimic acid =
V X OD535 =
33
3.3 × OD535 33
---- 0.1 × OD535 where V is the final volume of the test solution and optical density is measured in a l-cm light path. nArsenite-treated samples may be stored at least 5 hours at room temperature before the thiobarbituric acid step without a decrease in the final optical density reading.
354
[43c]
AROMATIC AMINO ACIDS
Reproducibility The average optical density for 0.03 micromole of shikimic acid in 12 assays at 3 different times was 0.296 with a standard deviation of + 0.013. Specificity The method is relatively nonspecific, since strong reactions are given by such compounds as deoxyribose, guanosine, guanylic acid, and adenosine. About 20% of the color is given by N-acetylneuraminic acid. Some preliminary purification is necessary before mixtures containing these materials and shikimic acid can be assayed. 5-Dehydroshikimic acid reacts in the assay but no color is obtained with shikimic acid 5-phosphate, unless it has been pretreated with intestinal phosphatase or hydrolyzed in 0.4 N HCI for 4 hours at 120 °.
[43c] S h i k i m a t e D e h y d r o g e n a s e
1-2 ( P e a s )
By DORIS BALINSKY and ALAN W. DENNIS
coo-
coo.H
0
OH
Dehydroshikimate
+ NADPH + H+
H ~ H HO
+
NADP +
OH
8hikimate
Assay Method The enzyme is assayed in the direction of shikimate oxidation by following the production of NADPH spectrophotometrically at 340 m/~.a Coupling the reaction with GSSG reductase, as previously described, 2 is not necessary, as NADPH production is linear for at least 5 minutes. 1Alternative names: EC 1.1.1.25 Shikimate: NADP oxidoreductase; 5-dehydroshikimic acid reductase. The preparation of this enzyme from E. coli was described in Vol. II [39]. The preparation of 5-dehydroshikimic acid was described in Vol. VI [74]. laA recent paper on Moraxella calcoacetica [J. L. Canovas, M. L. Wheelis, and R. Y. Stanier, EuropeanJ. Biochem. 3, 293 (1968)] has described an inducible shikimate dehydrogenase which is not pyridine nucleotide-linked but can be coupled to 2,6-dichlorophenolindophenol. Its Michaelis constant for shikimate is 20 times greater than is that of the NADP-linked enzyme in the same organism. ZB. D. Davis, C. Gilvarg, and S. Mitsuhashi, Vol. II [39]. SD. Balinsky and D. D. Davies, Biochem.J. 80,292 (1961).
AROMATIC AMINO ACIDS
[43a]
and finally with 500 ml of 5% unacidified ethanol. Shikimate-5-P was eluted with 2500 ml of 5% ethanol, 3 liters of 10% ethanol, and 2 liters of 25% ethanol. The fractions were combined and concentrated in a vacuum. The residual colorless syrup was diluted with water to 150 ml, and was found to contain 27 millimoles of shikimate-5-P. T h e solution was adjusted to pH 7 with NH4OH and treated with 100 ml of 0.5 M barium acetate. Absolute ethanol (950 ml) was added to give 80% (v/v) ethanol, and the solution was allowed to stand overnight at 0 °. T h e precipitate was collected by centrifugation, washed twice with 200 ml of 75% ethanol and once with 200 ml of absolute ethanol, and dried over P2Os under vacuum yielding 12.8 g (25 millimoles) of the barium salt as a white powder. Continued elution of the column with 10 liters of 25 % ethanol gave a slightly yellow syrup (assaying 7.9 g of shikimate5-P) from which was isolated 2.9 g of slightly tan barium salt. Both preparations of shikimate-5-P gave only one spot in descending chromatography on paper with butanol-I/acetic acid/water (100:6:2, v/v), 3 and with propanol-l/15 M N H 4 O H / H 2 0 (6:3:1, v/v). 4 However, a minor phosphorylated impurity appeared in paper electrophoresis for 1 hour at pH 5.5 and 900 volts (0.1 M sodium acetate buffer; length of paper 50 cm). An acid molybdate spray 4 was used to detect phosphate esters. Chromatography on Dowex-1 CI-X8 (200-400 mesh) at pH 9 (0.01 M Tris-Cl) with a lithium chloride gradient gave one symmetrical peak. The minor phosphate ester obtained in paper electrophoresis at pH 5.5 may have resulted from isomerization of the shikimate 5-phosphate to the 4-phosphate. 1 Hydrolysis of both barium salts with alkaline phosphatase gave rise to 1.0 mole of shikimate and 1.0 mole of Pl per equivalent weight of 500.1 sj. A. Fewster, Biochem.J. 85,388 (1962). ~;. S. Hanes and F. A. Isherwood, Nature 164, 1107 (1949).
[43b] Assay of Shikimic Acid By R. C. MILLICAN1 This assay2 depends on the treatment of shikimic acid with periodate 1Deceased-- March 7, 1964 2This procedure was described by R. C. Millican, Anal. Biochem. 6, 181 (1963). It is based on the procedure developed by L. Warren for the thiobarbituric acid assay of sialic acids [J. Biol. Chem. 234, 1971 (1959); Vol. VI [67]. Saslaw and Waravdekar (Biochim. Biophys. Acta 37, 367, 1960) have also published a method for shikimic acid determination, based on the thiobarbituric acid procedure.
[43b]
SHIKIMIC ACID ASSAY
353
in strong acid solution, followed by the reaction of the oxidation product with thiobarbituric acid. T h e chromogen is extracted into cyclohexanone to produce a stable color with a molar extinction of 33,000 at 535 m/~.
Reagents Shikimic Acid 4.28% Sodium metaperiodate in 9 M'H3PO4 10% Sodium arsenite in a solution of 0.5 M sodium sulfate-0.1 N sulfuric acid. 0.6% Thiobarbituric acid in 0.5 M sodium sulfate, adjusted to pH 7 with 5 N NaOH. Some material is insoluble, but this does not affect the color. Cyclohexanone
Procedure. To each sample containing up to 0.06 micromole of shikimic acid in a volume of 0.2 ml, 0.1 ml of periodate solution is added. The tubes are shaken and incubated at 80 ° for 5 minutes. At the end of the periodate oxidation 0.5 ml of arsenite solution is added and the tubes are shaken until the yellow-brown color disappears) Thiobarbituric acid solution, 2.5 ml, is added to each tube; the tubes are shaken, capped with a marble bead, and then heated in a vigorously boiling water bath for 15 minutes. During cooling the red color fades and the solution often becomes cloudy. This does not interfere with the final reading. One milliliter of this solution is transferred to another tube containing 1 m~ of cyclohexanone. The latter tube is ~ h a ~ n twice and centrifuged in a clinical centrifuge for 3 minutes. The clear upper cyclohexanone phase is red. The colors are very stable in the cyclohexanone phase. Optical densities are determined on the organic phase at 535 m g in a Beckman model,DU spectrophotometer with a 1-cm light path. Measurements are read against water. The color produced increases linearly between 0.01 and 0.06 micromole of added shikimic acid. The amount of shikimic acid present in a given sample can be determined from the following equation: micromoles shikimic acid =
V X OD535 =
33
3.3 × OD535 33
---- 0.1 × OD535 where V is the final volume of the test solution and optical density is measured in a l-cm light path. nArsenite-treated samples may be stored at least 5 hours at room temperature before the thiobarbituric acid step without a decrease in the final optical density reading.
354
[43c]
AROMATIC AMINO ACIDS
Reproducibility The average optical density for 0.03 micromole of shikimic acid in 12 assays at 3 different times was 0.296 with a standard deviation of + 0.013. Specificity The method is relatively nonspecific, since strong reactions are given by such compounds as deoxyribose, guanosine, guanylic acid, and adenosine. About 20% of the color is given by N-acetylneuraminic acid. Some preliminary purification is necessary before mixtures containing these materials and shikimic acid can be assayed. 5-Dehydroshikimic acid reacts in the assay but no color is obtained with shikimic acid 5-phosphate, unless it has been pretreated with intestinal phosphatase or hydrolyzed in 0.4 N HCI for 4 hours at 120 °.
[43c] S h i k i m a t e D e h y d r o g e n a s e
1-2 ( P e a s )
By DORIS BALINSKY and ALAN W. DENNIS
coo-
coo.H
0
OH
Dehydroshikimate
+ NADPH + H+
H ~ H HO
+
NADP +
OH
8hikimate
Assay Method The enzyme is assayed in the direction of shikimate oxidation by following the production of NADPH spectrophotometrically at 340 m/~.a Coupling the reaction with GSSG reductase, as previously described, 2 is not necessary, as NADPH production is linear for at least 5 minutes. 1Alternative names: EC 1.1.1.25 Shikimate: NADP oxidoreductase; 5-dehydroshikimic acid reductase. The preparation of this enzyme from E. coli was described in Vol. II [39]. The preparation of 5-dehydroshikimic acid was described in Vol. VI [74]. laA recent paper on Moraxella calcoacetica [J. L. Canovas, M. L. Wheelis, and R. Y. Stanier, EuropeanJ. Biochem. 3, 293 (1968)] has described an inducible shikimate dehydrogenase which is not pyridine nucleotide-linked but can be coupled to 2,6-dichlorophenolindophenol. Its Michaelis constant for shikimate is 20 times greater than is that of the NADP-linked enzyme in the same organism. ZB. D. Davis, C. Gilvarg, and S. Mitsuhashi, Vol. II [39]. SD. Balinsky and D. D. Davies, Biochem.J. 80,292 (1961).