1664
ANALYTICAL CHEMISTRY
the methoxy group by this method, whereas the Zeisel reaction is not specific for the methoxy group. LITERATURE CITED
Beroaa, bl., AKAL.CHEM.,26, 1970 (1954). Beyer, G., J . Assoc. Ofic. Agr. Chemists, 34, i 4 5 (1951). Boos, R. N., A s . 4 ~ CHEM., . 20, 964 (1948). Bricker, C E., and Johnson, H. R . , ISD. E m . CHEM.,ANAL. ED., 17, 400 (1945). ( 5 ) Bricker, C. E., and Roberts, K. H., AKAL.CHEM.,21, 1331 (1) (2) (3) (4)
(1949).
Bricker, C. E., and Vail, A4.H., Ibid., 22, 720 (1950). Eegriwe, E., 2. anal. Chem., 110, 22 (1337). 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,, Sei. A g r . , 22, GU (1341). (10) Mathers, A. P., “Report on Methanol Determination,” .innus1 Meeting of Assoc. Offic. Agr. Chemists, Opt. 12, 1954. (11) Xanju, D. R., and Norman, A. G., J. SOC.C‘hem. I n d . , 45, 337 (6) (7) (8)
(1926). (12)
Pavolini, T., and llalatesta, A.. Ann. chim. up&, 37,495 (194;).
RECEIVED for review February 18, 1955. Accepted July 26, 1955.
Spectrophotometric Method for Determining Hydroxylamine Reductase Activity in Higher Plants D. S. F R E A R
and R. C. BURRELL
D e p a r t m e n t o f Agricultural Biochemistry, The O h i o State University, Columbus, O h i o
A rapid, simple color test for hydroxylamine has been adapted for the quantitative determination of micromolar amountsof hydrovylaminein biological materials. The hydroxylamine reacts quantitatively w-ith an excess of 8-quinolinol to form the stable 5,8-quinolinequinone5-(8-hydroxy-5-quinolylimide). When measured spectrophotometrically at its absorption peak, 705 mp, this compound obeys Beer’s law over the range of 0 to 5 X millimole of hydroxylamine per nil. 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. 0.001iM solution. Reduced Diphosphopyridine Sucleotide Solution. Reduced diphosphopyridine nucleotide (Sigma Chemical Co.), 3 X 10-4M solution. Keep refrigerated. Phosphate Buffer Solution, p H 6.8. Adjust the p H of a 0.05M monobasic sodium phosphate solution to pH 6.8 by the addition of 0.05M dibasic sodium ohosnhate solution. Keea 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 p H 3.0 with 0,0lLVhydrochloric acid, and dilute to 1 liter. This solution contains 0.25 micromole of hydroxylamine per milliliter, and is stable for several days. ~
PROCEDURE
P
REVIOUS procedures for the determination of hydroxyl-
amine have been reported by Blom (W), Endres and IZaufniann ( 5 ) ,and Csaky ( 4 ) . These procedures involve the oxidation of hydroxylamine to nitrous acid, which is then determined colorimetrically by the Rider and bIellon (7) or S h i m (8) procedures. Although these methods are sensitive for the resulting nitrous acid formed, the oxidation of the hydroxylamine is neither specific nor simple. Xason and others (6) have demonstrated the presence and the requirements of hydroxylamine reductase in soybean leaves using Csaky’s 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 t o 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 mp (Figure 1).
(e)
Hydroxylamine Standard Curve. I n a 15 X 125 mni. teat 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 0.0511/1 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 sn-irl gently. Neut, add 1.0 ml. of the 1.OM sodium carbonate solution, shake vigorously, and stopper before placing in a boiling m-ater 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 D G spectrophotometer a t 705 mH using matched 1-em. Corex cuvettes. Carry out simultaneously a blank determination which contains everything but hydroxylamine and set a t 100% 2‘: Within this concentration range (0.00 to 0.25 micromole of hydroxylamine), the Beer-Lambert law is obeyed, giving R
REAGENTS
8-Quinolinol Solution. Dissolve 1.O 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.OM solution. Trichloroacetic .kcid Solution. Water solution, 12% by weight.
600
Figure 1.
700 WAVELENGTH I N MILLIMICRONS
800
Absorption spectrum of Indooxine
1665
V O L U M E 27, NO. 10, O C T O B E R 1 9 5 5 Effect of Hydroxylamine Concentration on Absorbance
'Tahle I. IIydro\ylaiiiine Concentration 10-2 p . U per LII.
x
Absorbance a t 705 niii I1 Ill IV
1
h\..
Beckman Dl27 spectrophotometer a t 705 mp. Carry out a blank determination which contains the enzyme system minus the hydroxylamine, and set a t 100% T (Table 11). To correct for any nonenzymatic hydroxylamine decomposition a t pH 6.8 in the presence of the plant tissue extract, concurrently carry out a determination containing boiled plant tissue extract. DISCUSSIOK
l'ablr 11. Time. Lliiiutes
?
Soybean Hydroxylamine Reductase Activity Plant Extract Absorbance a t 705 mfi 0 621 0 429 0 328
10 1.j
0 270
Boiled Plant E x t r a c t Absorbance a t 705 mfi 0.747 0.683 0 622 0 599
Hydroxylamine Loss Corrected, p.lf/lIl. 0.85 1 70 1.9.5 2.20
straight line Lvheii the :tbsort)ances are plotted against the hydroxylamine concentrations (Table I). The color, under these conditions, develops completely in 15 minutes and is sttitile for a t least another 30 minutes. Plant Tissue Extract. Grind 1 gram of leaves from young soyhean seedlings in a cold mortar with 1.0 gram of washed sand and 10.0 ml. of cold 0.05.M phosphate buffer pH 6.8. Strain the resulting brei through cheesecloth, centrifuge in the cold a t 20,000 times gravity for 10 minutes, decant, and use the supernatant solution directly for hydroxylamine reductase as Hydroxylamine Reductase Assay. I n a 15 X 125 IIIIIL test tube, place 1.0 ml. of the hydroxylamine standard solution (0.25 micromole of hydroxylamine), 1.0 ml. of the 0.U5M phosphate buffer, pH 6.8, 0.1 ml. of the 3 X 1OPAI reduced diphosphopyridine nucleotide solution, 0.1 ml. of the 0.001:lI manganese chloride solution, 0.3 ml. of the plant tissue extract, aiid 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 nil. of the trichloroacetic acid solution. F011o\v with the addition of 1.0 ml. of the 8-quinolinol solution, and swirl gently. A4dd1.0 ml. of the 1.0M sodium carbonate solution, shake vigorously, stopper, and place in a boiling vater bath for 1 minut'e. Centrifuge for 5 minutes a t 3000 times gravity, cool for 10 minutes, decant into matched 1-cm. Corex cuvettes, and read i n the
To ensure reproducible results, accurate volumetric measurements 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; consequent,ly, the test tubes must be shaken vigorously after the addition of the 1.0M sodium carbonate solution to ensure complete atmospheric oxidation. Above a pH of 6.8, hydroxylamine decomposes rapidly. Even at, p H 6.8, under the enzyme conditions specified here, t'here is a significant hydroxylamine decomposition in 10 minutes (Table 11). Rather large amounts of nitrous acid, glutathione, and ascorbic acid have been found to enhance t.he decomposition of hydroxylamine a t pH 6.8, but these concentrations are considerably in excess of any coiicentrations normally found in plant tissues. I n the crude plant tissue extracts used in these experiments there is also a slight loss of the hydroxlyamine bound to t,he large amount of precipitated protein. LITERATURE CITED
(1) Berg, R., and Becker, E., Ber., 73B, 172 (1940). (2) Blom, J., Biochem. Z., 194, 385 (1928). (3) Colter, J. S., and Quastel, J. H., Arch. Biochem., 27, 368 (1950). (4) Csaky, T. Z., Acta Chem. Scand., 2,450 (1938). ( 5 ) Endres, G., and Kauftnann, L., Ann., 530, 184 (1937). (6) Nason, A . , dkrsham, R. G. arid dverbach, B. C., Biochim. et B i o p h y s . Acta, 15, 159 (1954). (7) Rider, B. F., and Jlellon, 31. G., ISD. Eso. CmM., dsar.. ED., 18,96 (1946). (8) Shinn, AI. B., Ibid., 13, 3 3 (1941).
RECEIVED for review XIny 6 , 1955. hccepted July 18, 1955.
CRYSTALLOGRAPHIC DATA
99.
Hydrocortisone Acetate
Contributed by
JOHN W. SHELL, The
Upjohn Co., Kalamazoo, Mich.
CRYSTAL MORPHOLOGY Crystal System and-Class. Monoclinic, sphenoidal. Form and Habit. Tabular, shoiving forms ( 100), (0011, { 110 1, { O l l ! , and (031). Axial Ratio. a : b : c = 0.649:1:0.648. Beta Angle. 102.3'. X-RAYDIFFRACTION 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).
Principal Lines
Structural Formula for Hydrocortisone Acetate
of hydrocortisone acetate suitable for microscopic 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. RYSTALS
C
d 8.82 7.33 6.71 6.11 5.63 5.26 4.79 4.39 4.07 3.48
I/Il
d
I/II 8 4 4 3 4 1 1 1
1
