Vol. 67 PROTEINS OF NEPHROTIC SERUM AND URINE 445 Dr D. R. Stanworth for the carbohydrate stains and for an Grabar, P. & Williams, C. A. (1953). Biochim. biophy8. Acta, analysis in the ultracentrifuge. It is a pleasure to acknow- 10, 193. ledge the advice and encouragement of Professor J. R. Hardwicke, J. (1954a). Proc. R. Soc. Med. 47, 832. Squire. This work was done during the tenure of a grant Hardwicke, J. (1954b). Biochem. J. 57, 166. from the Medical Research Council. Ki6w, E. & Gronwall, A. (1952). Scand. J. clin. Lab. Invest. 4, 244. REFERENCES Ma, T. S. & Zuazaga, G. (1942). Industr. Engng Chem. (Anal.), 14, 280. Adair, G. S., Adair, M. E. & Greaves, R. I. N. (1940). Popjak, G. & McCarthy, E. F. (1946). Biochem. J. 40, 789. J. Hyg., (Camb., 40, 548. Rowe, D. S. (1956). J. Phy8iol. 134, 1P. Adair, G. S. & Robinson, M. E. (1930). Biochem. J. 24, 1864. Rowe, D. S. & Abrams, M. E. (1957). Biochem. J. 67, 431. Bayliss, L. E., Kerridge, M. T. & Russell, D. S. (1933). Schultze, H. E., Gollner, I., Heide, K., Schonenberger, M. & J. Phy8iol. 77, 386. Schwicke, J. (1955). Z. Naturf. 106, 463. Bourdillon, J. (1939). J. exp. Med. 69, 819. Squire, J. R., Blainey, J. D. & Hardwicke, J. (1957). Brit. Brewer, D. B. (1951). Proc. R. Soc. Med. 44, 557. med. Bull. 13, 43. Burtin, P. & Grabar, P. (1954). Sem. H6p. Paris, 30, 1. Tristram, G. R. (1953). In The Proteins, vol. 1A, p. 215. Campbell, P. N. & Stone, N. E. (1956). Biochem. J. 62, 9P. Ed. by Neurath, H. & Bailey, K. New York: Academic Charlwood, P. A. (1952). Biochem. J. 52, 279. Press. Flodin, P. & Porath, J. (1954). Biochim. biophys. Acta, 13, Wallenius, G. (1954). Acta Soc. Med., Upsalien, suppl. 4. 175. Wells, H. S. (1932). Amer. J. Phy8iol. 101, 409. Gell, P. G. H. (1955). J. clin. Path. 8, 269. Widdowson, E. M. (1933). Biochem. J. 27, 1321. The Transformation of Gallates into Ellagate BY D. E. HATHWAY The Briti8h Leather Manufacturers' Research Association, Milton Park, Egham, Surrey (Received 1 April 1957) Herzig, Pollak & Bronneck (1908) demonstrated products of reaction. In this study, 4:5:6:4':5':6'- that aeration of an ammoniacal solution of ethyl hexahydroxydiphenic acid was shown to be the gallate (I) yielded ellagic acid (V), and recently chemical precursor of ellagic acid, and gallic acid humic acid and a trace of hydrogen peroxide were the chemical precursor ofthe humic acidby-product. also found (Hathway, 1957) amongst the ultimate It is now suggested that the principal reaction may HO 00 HO H ~CO2R ri HO~~~~~~~~~~~OO Gallic acid (R =H) JOHOs (I), where R =Et. 00 (II), where R =Me. Ellagic acid (III), where R =#-D-glucose-l-. (V) HO HICOt CO-C wCH*C02HHO, HC0H HO HO 00 ~~~~~~HCOC-0-OH H HO 0 H HOt XC~~~O*OCHI 00-OHt-O H CheuinIi CHcOiH hObla0OIO-0-OH1-OHci H O-HOO 00N H HO ~~HO* O H oc ~~~OH HO OTH HO OH HO HO-O O LH2~ ~ ~ HLJ,H2 OCheblulinic acid Chebulagic acid (IV) (VI) 446 D. E. HATHWAY I957 have biological implications. Quantitative aspects Oxidation procedure. In a typical experiment, 0-5 m-mole ofthe transformation in vitro ofplant metabolites of of finely divided substrate was dissolved in 150 ml. of type (I)-(IV) into ellagic acid which have been buffer which had previously been equilibrated at 200, and explored under physiological conditions are re- the unit was connected in series with the air supply. At the end of the reaction period the flask was detached from the ported in the present communication. train, and the reaction was arrested by the acidification of the reaction mixture with conc. HCI. Ellagic acid was co- MATERIALS AND METHODS agulated, and filtered in a 4G sintered-glass crucible, which Melting points were determined on a Kofler block. Specific was subsequently washed with hot water and ethanol rotations were determined for the D line of sodium, a 1 dm. successively, and dried at 1000 to constant weight. When the microtube being used. Five pairs of readings with the acidified reaction mixture contained insoluble enzyme pre- solutions and five pairs with a solvent blank were taken in paration, the supernatant was removed from the sediment each case. Temperature, where not recorded, was 20-25°. in the centrifuge, and the sediment was boiled with a 10 ml. Paper chromatography was carried out at 23±20 in all- portion of pyridine. The pyridine extract was filtered from glass apparatus. Chromatograms were dried at room cellular debris, which was subsequently washed with three temp. unless otherwise stated. A Hanovia mercury arc further 10 ml. portions of boiling pyridine. The combined fitted with a Wood's glass filter was used to examine chro- pyridine extract and washings were diluted with water and matograms for fluorescent zones. acidified with conc. HCI. After coagulation, the ellagic acid Ethyl and methyl gallates were prepared by Fischer- precipitate was collected as previously described. This Speier ethylation and methylation of the free acid, and they separation afforded reproducible results. were crystallized from aq. ethanol and aq. methanol Autoxidation and enzymic-oxidation experiments in- respectively and dried over P.O. at 1200/0.01 mm. Hg. volving glucogallin and chebulinic acid were performed Ethyl gallate formed rhombs, m.p. 1550; methyl gallate under similar conditions, chebulinic acid being extracted formed from ellagic acid precipitate with a large volume of acetone. rhombs, m.p. 198-199°. the Glucogallin (,-D-glucose-l-gallate) was synthesized from Aerobic oxidation of gallate in presence of ascorbic acid ,-D-glucose-2:3:4:6-tetra-acetate and galloyl chloride tri- was carried out similarly. In the attempts at coupled oxida- acetate, according to Fischer & Bergmann's method (1918). tion, methyl gallate (0-5 mm), ascorbic acid (0.5 mM) and Deacetylation of the hepta-acetate, m.p. 1250, [I]D- 24' copper (2-5pg./ml.) were present in equilibrated phosphate (c, 1-0; tetrachloroethane), by Zemplen ammonolysis under buffer, pH 7 4, and the experiment was repeated in the H., gave free glucogallin which crystallized from 80% presence of 3 x 10-2 E.U. of mushroom polyphenoloxidase. ethanol, forming small prisms, m.p. 211-212°, [M]D -240 Oxidation of ascorbic acid. Ascorbic acid (35.2 mg.) was (c, 1-0; water). dissolved in 200 ml. of 0-75M-NaHCO3, pH 7*9, which was aerated at 200. were withdrawn at 15 min. Chebulinic acid nonahydrate, [z]D + 650 (c, 1-0; aq. Samples (1 ml.) ethanol) was isolated from the dried fruit (myrobalans) of intervals, acidified to Congo red with 20 % (w/v) trichloro- Terminalia chebula, and was crystallized from aq. acetone acetic acid and titrated immediately with 0-02 mM-2:6- (Freudenberg & Frank, 1927). The preparation ran as a dichlorophenolindophenol. single spot on a two-way chromatogram, in which N-acetic acid and butan-2-ol-acetic acid-water (14:1:5, by vol.) Identification of the productfrom the autoxidations were used as solvent systems. and enzymic oxidation8 L-Ascorbic acid (Biochemicals Roche) had m.p. 188-190°, Paper chromatography. Spots (5uA.) of an 0-2% solution [a]D + 480 (c, 1-0; methanol). of the product in pyridine, and marker spots (5 ,ul.) of 0-2 % Soluble lyophilized mushroom polyphenoloxidase (L. solutions of authentic ellagic acid in pyridine, and of Light and Co.) had a purpurogallin number (P.s.) of 0-15. galloflavin and isogalloflavin in methyl Cellosolve (2- P.N. represents the no. of milligrams of purpurogallin formed methoxyethanol), were applied to starting lines 4 cm. from from pyrogallol in 5 min. at 20°/mg. dry wt. of enzyme the lower edge of sheets of Whatman filter paper no. 1, of preparation (Keilin & Mann, 1938). length 57 cm. Single-way ascending chromatography was Acetone-dried powder of Psalliota campestris. Freshly effected with benzyl alcohol-tert.-butyl alcohol-propan-2- picked mushrooms (300 g.) were sliced and put into ice-cold ol-water (3: 1: 1: 1, by vol.) containing 1.8 % (w/v) of formic acetone (2 1.) and homogenized in a macerator. The homo- acid (Stark, Goodban & Owens, 1951). Papers were irrigated genate was filtered on sintered glass, and the solid (20 g.) for 20-24 hr., and dried at 60-700. Single-way chromato- was immediately washed five times with 500 ml. portions of graphy of the product and of authentic ellagic acid was also ice-coldacetone and dried over paraffin wax at 00/1 mm. Hg. effected in aq. 60% (w/v) formamide buffered at pH 3-5 A Hilger Uvispek spectrophotometer was used for the with formic acid (Hathway, 1956a). Ellagic acid was P.N. determination by Keilin & Mann's (1938) method. This recognized by its distinctive soft violet fluorescence in u.v. powder (2.8 enzyme units) had a P.N. of 0-14. One enzyme light, changing to pale yellow on exposure to NH3 vapour. unit (xi.u.) corresponds to the quantity of enzyme which Derivative formation. A filtered solution of the reaction produces 1 g. of purpurogallin in 5 min. at 200. product in pyridine was regenerated by acidification with Incubation unit. A 500 ml. Erlenmeyer flask equipped conc. HCI. The precipitate was removed, dried at 1000, and with a B24 standard ground-glass socket to receive a gas refluxed with acetic anhydride in the presence ofa few drops wash-bottle head (MF 28/3, Quickfit and Quartz Ltd., of conc. H2SO4 as catalyst. Ellagic acid tetra-acetate London) with an inlet reaching to within 5 mm. of the crystallized from a large volume of acetic anhydride, bottom of the flask, was immersed in a thermostat at 200.