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
ANALYTICAL CHEMISTRY
1666 OPTIC*L
Refrm Y
= 1.6:
ODtiC
Figure 2.
Hydrowrtisone a c e t a t e growing from melt
Seeded at edge (dark area) with cqsfaln fmm acetonewater
Fmm acetone-water
The specific rotation, [elD,of the hydrocortisone acetate used far these determinations is 164" (dioxane). Typical crystals from acetone-water are shown in Figure 1, 75X. Figure 2 shows the crystals from fusion after seeding a t the edge of the cover glass (dark ares), 150X. The melting point is 224' C.; the melt does not recrystallize unless seeded. Orthographic projections are shown in Figure 3. The unstable solvate, obtained when hydrocortisone acetate is crystallized from dimethylformamide, is partially characterized by the following data. Because of this compound's instability, further characterization has not been made.
+
Crystal System and Class. Orthorhombic, rhombic dipyramidal.. Forms Present. Unit prism, { l l O ] ; brachy pinacoid, lOl01; macro dome, [ 101 1. Axial Ratio. a:b:c = 0.614:1:0.312. Cell Dimensions. a = 15.2 A,; b = 24.74 A.; e = 7.71 A.
II
Figure 3. Orthographic projection of hydrocortisone aoetate
Refractive Index (5893 A,, 25" C.). Optic Sign. Positive. Dispersion. Y > 7, very strong. Optic Axial Angle, Very small. Optic Orientation. X = e, Y = a.
01
= 1.558
Coli~n~smroaa of crystsllograDhio data for this section shonld be sent to Walter C. MeCrone. Analytioal Research. Armour Researoh Foundation. Illinois Institute of Teohndogy, Cliieapo 16, Ill.
