ANALYTICAL
1664
the methoxy group by this method, whereas the Zeisel reaction is not specific for the methoxy group. LITERATURE
CITED
Beroza, M., Anal. Chem., 26, 1970 (1954). Beyer, G., J. Assoc. Offic. Agr. Chemists, 34, 745 (1951). Boos, R. N., Anal. Chem., 20, 964 (194S). Bricker, C. E., and Johnson, H. R., Ind. Eng. Chem., Anal. Ed., 17, 400 (1945). (5) Bricker, C. E., and Roberts, K. H., Anal. Chem., 21, 1331 (1949). (1) (2) (3) (4)
CHEMISTRY
(6) Bricker, C. E., and Vail, A. H., Ibid., 22, 720 (1950). (7) Eegriwe, E., Z. anal. Chem., 110, 22 (1937). (8) Hawley, L. F., and Wise, L. E., “Chemistry of Wood,” p. 263, Chemical Catalog, New York, 1926. (9) Manning, K. R., and DeLong, W. A., Sci. Agr., 22, 69 (1941). (10) Mathers, A. P., “Report on Methanol Determination,” Annual Meeting of Assoc. Offic. Agr. Chemists, Oct. 12, 1954. (11) Nanju, D. R., and Norman, A. G., J. Soc. Chem. Ind., 45, 337
(1926).
(12) Pavolíni, T., and Malatesta, A., Ann. chim.. appl., 37, 495 (1947). Received for review February 18, 1955.
Accepted July 26, 1955.
Spectrophotometric Method for Determining Hydroxylamine Reductase Activity in Higher Plants D. S. FREAR and R. C. BURRELL
Department of Agricultural Biochemistry, The Ohio State University, Columbus, Ohio
A rapid, simple color test for hydroxylamine has been of microadapted for the quantitative determination molar amounts of hydroxylamine in biological materials. The hydroxylamine reacts quantitatively with an excess of 8-quinolinol to form the stable 5,8-quinolinequinone5-(8-hydroxy-5-quinolylimide). When measured spectrophotometrically at its absorption peak, 705 mg, this compound obeys Beer’s law over the range of 0 to 5 X 10 "2 millimole of hydroxylamine per ml. of solution. The procedure has been applied successfully for the determination of hydroxylamine reductase activity in soybean leaves.
Manganese Chloride Solution. Manganese chloride, c.p. 0.0013/ solution. Reduced Diphosphopyridine Nucleotide Solution. Reduced diphosphopyridine nucleotide (Sigma Chemical Co.), 3 X 10-43/ solution. Keep refrigerated. Phosphate Buffer Solution, pH 6.8. Adjust the pH of a 0.053/ monobasic sodium phosphate solution to pH 6.8 by the addition of 0.053/ dibasic sodium phosphate solution. Keep refrigerated. Hydroxylamine Standard Solution. Dissolve 0.0695 gram of dry, recrystallized hydroxylamine hydrochloride, c.p., in water and dilute to 1 liter. Take a 250-ml. aliquot of this solution, adjust to pH 3.0 with 0.0LV hydrochloric acid, and dilute to 1 liter. This solution contains 0.25 micromole of hydroxylamine per milliliter, and is stable for several days. PROCEDURE
procedures for the determination of hydroxylamine have been reported by Blom {2), Entires and KaufPREVIOUS mann (5), and Csaky (4). These procedures involve the oxidation of hydroxylamine to nitrous acid, which is then determined colorimetric ally by the Rider and Mellon (7) or Shinn (8) procedures. Although these methods are sensitive for the resulting nitrous acid formed, the oxidation of the hydroxylamine is neither specific nor simple. Nason and others (6) have demonstrated the presence and the requirements of hydroxylamine reductase in soybean leaves using Csaky’s (4) method for the determination
of hydroxylamine. Colter and Quastel (3) have recently reported a manometric procedure for the determination of hydroxylamine, which depends upon the oxidation of hydroxylamine by manganese dioxide to produce nitrous oxide. This method, however, is not very sensitive and is also rather involved. Berg and Becker (1) have reported a very sensitive and specific qualitative color test for hydroxylamine. This test has now been applied quantitatively to biological materials for the determination of micromolar quantities of hydroxylamine. The hydroxylamine reacts quantitatively with an excess of 8-quinolinol in the presence of ethyl alcohol and sodium carbonate to form the stable 5,8-quinolinequinone-5-(8-hydroxy-5-quinolylimide) designated as Indooxine. This compound exhibits a very prominent adsorption peak at 705 mg (Figure 1).
Hydroxylamine Standard Curve. In a 15 X 125 mm. test tube place up to 1.0 ml. of the hydroxylamine standard solution (0.00 to 0.25 micromole of hydroxylamine), 1.0 ml. of the 0.05M phosphate buffer, pH 6.8, and water to bring the volume to 2.8 ml. Add 0.2 ml, of the trichloroacetic acid solution. Follow with 1.0 ml. of the 8-quinolinol solution and swirl gently. Next, add 1.0 ml. of the 1.03/ sodium carbonate solution, shake vigorously, and stopper before placing in a boiling water bath for 1 minute to develop the green color. On removal from the water bath, cool for 15 minutes, and then read in the Beckman DU spectrophotometer at 705 mg using matched 1-cm. Corex cuvettes. Carry out simultaneously a blank determination which contains everything but hydroxylamine and set at 100% T. Within this concentration range (0.00 to 0.25 micromole of hydroxylamine), the Beer-Lambert law is obeyed, giving a
REAGENTS
8-QuinolinoI Solution. Dissolve 1.0 gram of 8-quinolinol (Eastman Kodak Co.) in 100 ml. of absolute ethyl alcohol. Keep
tightly stoppered.
Sodium Carbonate Solution. Sodium carbonate, c.p., 1.03/ solution. Trichloroacetic Acid Solution. Water solution, 12% by weight.
MILLIMICRONS Figure
1.
Absorption spectrum of Indooxine
VOLUME
10, OCTOBER
NO.
2 7,
Hydroxylamine
Absorbance at 705
Concentration
t.M per Ml.
1
0.146
1
0.305
2
0.442
3
0.599
4
0.720
5
II.
Time.
Minutes
1665
Effect of Hydroxylamine Concentration on Absorbance
Table I.
Table
1955
µ
II
III
IV
Av.
0.154 0.304
0.151
0.153 0.314
0.151
0.749
0.596 0.743
0.315 0.449 0.592 0.742
0.451 0.599
0.762
Plant Extract Absorbance at 705
µ
0
0.621
10
0.328 0.270
Boiled Plant Extract Absorbat 705
ance
1.3
Activity
Hydroxylamine
Loss Corrected,
µ
0.747 0.683 0.622 0.599
0.429
0.310 0.450
0.457 0.504
Soybean Hydroxylamine Reductase '2
Beckman DU spectrophotometer at 705 µ. Carry out a blank determination which contains the enzyme system minus the hydroxylamine, and set at 100% T (Table II). To correct for any nonenzymatic hydroxylamine decomposition at pH 6.8 in the presence of the plant tissue extract, concurrently carry out a determination containing boiled plant tissue extract.
µ. // 1.
0.85 1.70 1.95
2.20
straight line when the absorbances are plotted against the hydroxylamine concentrations (Table I). The color, under these conditions, develops completely in 15 minutes and is stable for at least another 30 minutes. Plant Tissue Extract. Grind 1 gram of leaves from young soybean seedlings in a cold mortar with 1.0 gram of washed sand and 10.0 ml. of cold 0.05 7 phosphate buffer pH 6.8. Strain the resulting brei through cheesecloth, centrifuge in the cold at 20,000 times gravity for 10 minutes, decant, and use the supernatant solution directly for hydroxylamine reductase assay. Hydroxylamine Reductase Assay. In a 15 X 125 mm. test tube, place 1.0 ml. of the hydroxylamine standard solution (0.25 micromole of hydroxylamine), 1.0 ml. of the 0.053/ phosphate buffer, pH 6.8, 0.1 ml. of the 3 X 10_43/ reduced diphosphopyridine nucleotide solution, 0.1 ml. of the 0.0013/ manganese chloride solution, 0.3 ml. of the plant tissue extract, and water to make a final volume of 2.8 ml. Incubate in a 30° C. water bath. Stop the enzyme action by the addition of 0.2 ml. of the trichloroacetic acid solution. Follow with the addition of 1.0 ml. of the 8-quinolinol solution, and swirl gently. Add 1.0 ml. of the 1.03/ sodium carbonate solution, shake vigorously, stopper, and place in a boiling rvater bath for 1 minute. Centrifuge for 5 minutes at 3000 times gravity, cool for 10 minutes, decant into matched 1-cm. Corex cuvettes, and read in the
DISCUSSION
To
ensure
reproducible results, accurate volumetric
measure-
ments of all additions, especially the hydroxylamine, along with
identical experimental procedure and experimental conditions are absolutely essential. The last step of the reaction of hydroxylamine with 8-quinolinol to form Indooxine is an oxidative step; consequently, the test tubes must be shaken vigorously after the addition of the 1.03/ sodium carbonate solution to ensure complete atmospheric oxidation. Above a pH of 6.8, hydroxylamine decomposes rapidly. Even at pH 6.8, under the enzyme conditions specified here, there is a significant hydroxylamine decomposition in 10 minutes (Table II). Rather large amounts of nitrous acid, glutathione, and ascorbic acid have been found to enhance the decomposition of hydroxylamine at pH 6.8, but these concentrations are considerably in excess of any concentrations normally found in plant tisIn the crude plant tissue extracts used in these experiments sues. there is also a slight loss of the hydroxlyamine bound to the large amount of precipitated protein. LITERATURE (1) (2) (3) (4)
(5) (6) (7)
(8)
CITED
Berg, R., and Becker, E., Ber., 73B, 172 (1940). Blom, J., Biochem. Z„ 194, 385 (1928). Colter, J. S., and Quastei, J. H., Arch. Biochem,, 27, 368 (1950). Csaky, T. Z., Acta Chem. Scand., 2, 450 (1948). Endres, G., and Kaufmann, L., Ann., 530, 184 (1937). Nason, A., Abraham, R. G. and Averbach, B. C., Biochim. et Biophys. Acta, 15, 159 (1954). Rider, B. F., and Mellon, M. G., Ind. Eng. Chem., Anal. Ed., 18,96 (1946). Shinn, . B., Ibid., 13, 33 (1941).
Received for review May 6, 1955.
Accepted July 18, 1955.
CRYSTALLOGRAPHIC DATA 99.
Hydrocortisone Acetate
Contributed by JOHN W. SHELL, The Upjohn Co., Kalamazoo, Mich. O II
H2C—O—C—CHs I
c=o
Crystal Morphology Crystal System and Class. Monoclinic, sphenoidal. Form and Habit. Tabular, showing forms {1001, (001), {110j, {Oil}, and {031}. Axial Ratio. a:b\c 0.649:1:0.648. Beta Angle. 102.3°. X-Ray Diffraction Data Cell Dimensions, a 8.85 A.; b 13.64 A.; c 8.84 A. Formula Weights per Cell. 2. Formula Weight. 404.49; 404.57 (x-ray). Density. 1.289 (flotation); 1.288 (x-ray). =
=
Structural
Formula for Hydrocortisone Acetate
of hydrocortisone acetate suitable for microscopic Crystals and x-ray study are readily obtained from an acetone-water solution. This compound is only slightly soluble in most organic solvents, except dimethylformamide, in which it is exceedingly soluble. There is no evidence of polymorphism, although an unstable solvate does form from dimethylformamide solutions.
=
=
Principal Lines d
8.82 7.33 6.71
I/h
d
I/h
6.11 5.63 5.26
4 9 5 5
3.15 2.82 2.67
8 10
2.45
4
2.18
1 1
4.39 4.07
5 4 7
4.79 3.48
2.59 2.27
2.09 2.04
8
4 4 3
1
1
