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portance and may be accounted for by variations in structure or minor impurities. None of the samples gave a positive ’flame test for sodium.
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Narain’s results. But even if the oxidases were not permanently inactivated by boiling, t h e matter of their classification still depends entirely on t h e definition of “enzymes.” Schmidt,l Gramenitzki,2 Mellanby SUMMARY and W ~ o l l e y ,report ~ t h a t under certain conditions, I-The relative rates of moisture absorption of three trypsin, taka-diastase, a n d other enzymes can be samples of black powder in a saturated atmosphere at boiled, a n d trypsin be heated in glycerol t o 292O, 2 j O have been determined. without pqrmanently losing their activity, and yet no2-The presence of potassium perc-hlorate up t o j body has ceased t o classify them as enzymes. If we per cent in mixtures with potassium nitrate has been accept t h e definition of B a y l i ~ s who , ~ says t h a t , “ E n shown t o have only slight effect on t h e rate of mois- zymes are merely a particular class of catalysts, cont u r e absorption. sidered for convenience apart, owing t o t h e fact t h a t 3-Charcoal has been shown t o contribute t o t h e they are produced b y living organisms and are, f o r hygroscopic property of black powder b u t in much t h e most part, of unknown chemical constitution,’’ then t h e resistance t o heat is not a sure criterion f o r smaller degree t h a n nitrate. classification. I t is true t h a t oxidases differ from other EXPLOSIVES CHEMICAL LABORATORY BUREAUOF MINES,PITTSBURGH. PA enzymes also in not being specific, but even this is no reason for separate classification under Bayliss’s definition. THE COLOR CHANGES OF SUGAR-CANE JUICE AND Narain in t h e same paper also publishes some obTHE NATURE OF CANE TANNIN’ servations on the polyphenol of the cane. He states Py F W. ~ Z R E A N t h a t on account of its behavior with lime water i t Received M a y 2 , 1919 must belong t o the oak tannin group and not t o t h e A former article on this subject2 dealt with the r81e gallotannic acid group, b u t t h a t some of its reactions of oxidases and of iron in t h e color changes of sugar- resemble those of pyrogallol. He was unable t o isocane juice. The third factor known t o be involvedin late pyrocatechin from t h e cane. This polyphenol, t h e reactions is a water-soluble polyphenol which gives according t o Miss Wheldale,6 is t h e cause of t h e guaiac a green coloration with -.ferric salts. The previous reaction given by a number of plants. The author’s literature regarding t h e whole subject was discussed experiments fully confirm Narain’s claims on t h i s in t h e paper referred to, but since its publication there point. Miss Wheldale’s supposed pyrocatechin was has been noticed a very interesting and valuable con- most probably some other substance, because she retribution, overiooked before, on t h e oxidases of t h e ports finding it after treatment of t h e plant extract sugar cane, by Ramji NaraineS His studies were made with chloroform, in which solvent pyrocatechin is more particularly from t h e standpoint of t h e plant appreciably soluble. Dekker6 also expresses serious. physiologist, t h e principal object being t h e discovery doubfs concerning t h e alleged occurrence of pyroof possible connections between oxidase activity and catechin in plants. The absence in sugar cane of t h e formation of carbohydrates in t h e plant. He ob- pyrocatechin was proven b y us by digesting sliced tained positive reactions for a laccase and for per- cane tops, which give a strong reaction with ferric salts, oxidase, but did not find tyrosinase, evidently because with benzene in a glass-stoppered bottle. The benzene he did not employ t h e specific reagent consisting of extract was evaporated, t h e residue treated with water, p-cresol and glycocoll. Narain also reports some filtered, and t h e filtrate tested for pyrocatechin. I t observations regarding t h e thermostability of cane did not even give t h e iron reaction. oxidase. He found t h a t his oxidase preparations could I n our further investigations on the nature of t h e be boiled vigorously for I j min. without t h e permanent cane polyphenol, we first tried t o prepare extracts loss of their effect. Their activity was found t o be rich in this substance b y dropping sliced cane tops, greatly impaired directly after boiling, b u t it reappeared almost undiminished after cooling. The boil- and eyes cut from t h e cane, into alcohol. This was done during t h e grinding season, and we hoped t o work ing for I j min. could even be repeated with t h e same up these extracts after t h e end of t h e campaign. But effect as before. Like observations were made b y him i t was found t h a t , in spite of t h e high percentage of upon treating t h e oxidase preparations with hydrogen alcohol which was used t o weaken enzyme activity, sulfide. Similar results had been previously obtained, the extracts darkened very much while being kept, by Euler and Bolin, and b y others, b u t it appears t h a t and we concluded t h a t a large part of t h e polyphenol the action of heat on oxidases depends largely on their originally present must have been oxidized. After state of purity. Narain draws t h e conclusion t h a t a number of unsuccessful attempts t o prevent this oxioxidases can no longer be classed as enzymes, because dation, we finally adopted t h e method of slicing cane they do not show t h e characteristic thermolability of these substances. Neither Browne nor Raciborski tops directly into boiling water, continuing t h e boiling for 5 t o I O min., a n d then pressing t h e juice out rapidly report t h e return of t h e activity, of cane oxidase after 1 2. Ohysiol. Chem., 67, 314. boiling, and our own experiments did not confirm 2 I b i d , 69, 286. 1 Presented at the 56th Meeting of the American Chemical Society, Cleveland, Ohio, September 9 to 13, 1918. c *THISJOURNAL, 10 (1918), 814. A g y . J . I n d i a , 1918. Science Congress hrumber, 47.
8
4 6
6
Bayliss, “The Nature of Enzyme Action,” 3rd Ed.. p 9 7 . LOC.cit., pp, 1 1 , 139 Proc. R o y . S O C 84B , (1911), 121. “De Looistoffen,” 1, p. 209. @
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in a fruit press. The extract had only a faint green color, from small quantities of iron dissolved from the press. T h e further treatment was based on the fact, ascertained by special tests, t h a t the polyphenol is not precipitated by lead nitrate, but practically completely by neutral lead acetate. We, therefore, added a t once, t o t h e still hot extract a slight excess of lead nit r a t e solution. This caused a heavy precipitate which gave no reaction with ferric salts and consisted largely of lead sulfate, phosphate, organic lead salts, and other organic matter. The precipitate was filtered off, and the filtrate treated with neutral lead acetate solution in slight excess. The filtrate from this gave only a very faint iron reaction. The precipitate was suspended in water and decomposed with hydrogen sulfide. After filtering off t h e lead sulfide, t h e solution, which showed only a slight yellow color, was rapidly concentrated under diminished. pressure, and finally brought t o dryness in a vacuum desiccator over sulfuric acid. The residue was a buff-colored, amorphous mass and had an astringent, but. a t t h e same time very acid taste. The product obtained was further purified by treating in an extraction apparatus for 1 2 hrs. with anhydrous ether, in which the polyphenol is almost insoluble, The ether extract, upon evaporation, left a considerable quantity of a crystalline substance which would has7e contained any pyrocatechin present in t h e crude product. It showed a slight iron reaction, and was, therefore, extracted with benzene. The benzene was evaporated off, and a water extract of the residue tested for polyphenols with iron solution. The result was absolutely negative, a n d the reaction obtained before with t h e ether extract must have been due t o small quantities of t h e polyphenol itself, dissolved by ether. T h a t part of t h e ether-soluble material which was n o t dissolved by benzene was suspected t o consist of aconitic acid. This acid had been previously found in cane juice by Behr' and Szymanski,2 and was proven b y Yodera t o be t h e principal organic acid in sugar cane. Aconitic acid is precipitated by lead acetate, but not by lead nitrate. The crude acid obtained from t h e ether extract mentioned above was first converted into the acid ammonium salt, and this crystallized. The water solution of this salt was acidified, extracted with ether, and the ether evaporated. The acid was then once more recrystallized, and t h e resulting crystals showed under t h e microscope the characteristic appearance of aconitic acid.' The melting point was found t o be 178O, while t h e pure acid, made synthetically, melts a t 191 O . But Behr found for aconitic acid from cane a melting point of 171 t o 173', and SzymanThe acid was further identified by analysis ski 167'. of the silver salt, which contained 65.28 per cent silver, against 6 5.45 per cent calculated. The polyphenol preparation, after t h e removal of ether-soluble substances, was digested with alcohol, a n d t h e insoluble gummy substances filtered off. T h e solution was again evaporated under diminished Ber., 1 ( 1 8 7 7 ) , 351 a Be7 Versuchsst. West-Jnva, 2 (1896),25. 8 T H IJOURNAL, ~ 3 (191l ) , 640. 1
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pressure, and finally dried in a vacuum desiccator over sulfuric acid. The material obtained in this way was still amorphous, and had the same appearance as t h e original preparation before treatment with ether and alcohol. A number of tests were first made t o see whether t h e polyphenol belonged t o the tannins, or was of simpler constitution. It was found t o be soluble in water, alcohol, acetone, and ethyl acetate, but practically insoluble in ether, benzene, and chloroform. A I per cent sollttion of the material gave a precipitate with acidified gelatin-salt solution. This reaction is generally considered characteristic of the tannins, and is not given by simpler polyphenols, except by gallic acid in the presence of gum arabic. The delicate reaction of Allen, with an ammoniacal solution of potassium ferricyanide, was also positive. Lime water gave a t the first moment an olive coloration which quickly changed t o yellowish brown. Upon standing, a brown precipitate settled out. Bromine water gave a t once a yellow precipitate, which darkened and increased in quantity on standing. One per cent ferric alum solution gave a pure green color, which on careful addition of sodium bicarbonate solution first darkened, then changed t o bluish purple, and finally to purple-red. ' Ferric chloride and sodium acetate also gave a green color, but upon slowly adding dilute bicarbonate solution, t h e color first changed t o greenish blue, and then through purple t o purplish red. The exact color obtained depends entirely on t h e hydrogen ion concentration, and this shows t h a t t h e differentiation between tannins on the basis of their iron reaction is quite arbitrary, because t h e color may be green or blue according t o the reaction. Sodium nitrite plus a few drops of N / I O hydrochloric acid produced a reddish brown color, which did not change further. One per cent copper sulfate solution gave a precipitate which dissolved in ammonia with an olive color. Stannous chloride in concentrated hydrochloric acid developed on standing a pink coloration. With solid sodium sulfite no reaction was obtained, and pine wood moistened with hydrochloric acid gave only a faint bluish green color. This behavior towards reagents would make it ap. pear t h a t t h e cane polyphenol belongs t o Class 101 in Proctor's classification.' For t h e purpose of further identification the effect of heat, of dilute acids, a n d of potash fusion on the polyphenol was investigated. ACTION OF HEAT-one-half gram of t h e polyphenol preparation in 5 cc. of glycerol was gradually heated t o zoo', and t h e heating continued a t this temperature for 2 5 min. After cooling, t h e mixture was diluted with water and shaken out several times with ether. The residue left after evaporating the ether was found t o be soluble in benzene. I t gave a fine green color with ferric chloride, changing t o purple and red upon addition of sodium bicarbonate, and a pine splinter moistened with hydrochloric acid developed a bluish green color. No characteristic reaction was obtained with lime water. The melting point was found t o be 1
J . SOC.Ckem I d . , 13 (1894), 487
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9 8 ’. It was, therefore, pyrocatechin, and pyrogallol sulfuric acid and filtered from the potassium sulfate, was evidently not formed by heating the raw material. which crystallized out on standing. The liquid was E F F E C T OF ACIDS-This was investigated by heat- almost neutralized with sodium bicarbonate, shaken ing 2 g. of the original polyphenol preparation with I O O out several times with ether, the ether evaporated, cc. of 2 per cent hydrochloric acid under reflux for and the residue redissolved in water. The product was 2 * / 2 hrs. in a vigorously boiling water bath. Upon further purified by precipitating with neutral lead cooling, a small quantity of a reddish precipitate acetate, decomposing the precipitate with hydrogen settled .out, which was filtered off. It dissolved rea’dily sulfide, filtering off the lead sulfide, and evaporating in cold alcohol, and was reprecipitated from the solution the solution. I n this way a mass of fine, silky needles by adding water acidulated with hydrochloric acid. was obtained, slightly tinged with brown. They were It was, therefore, a phlobaphene, no ellagic acid being soluble in water, alcohoi, and ether, but not in benzene. formed. The filtrate from the phlobaphene was shaken They gave all the characteristic reactions of protoout several times with ether, and the ether evaporated. catechuic acid described above, and the melting point A considerable residue remained which was insoluble was found t o be 194’. in benzene. It was taken up in water and the soluThe filtrate from the lead acetate precipitate was tion filtered. I t gave a precipitate with neutral freed from lead and then tested for phloroglucin, lead acetate, soluble in acetic acid. The filtrate from but none could be found. The test with pine wood and t h e lead precipitate gave no precipitate with lead hydrochloric acid produced a bluish green color, due subacetate. Ferric alum solution produced a pure evidently to some protocatechuic acid, w.hich had not green color, which, on addition of sodium bicarbonate, been precipitated by t h e lead acetate. first changed t o blue and then t o a beautiful purplish The original product resulting after t h e potash fusion, red. Ferrous sulfate in neutral solution gave a fine when acidified with sulfuric acid, developed a strong violet color. Silver nitrate solution was reduced on odor of acetic acid. A part of the liquid was, therefore, warming, and ammoniacal silver solut.ion in the cold. distilled with steam, and a silver salt prepared from the Pine wood moistened with hydrochloric acid was distillate. Evidently small quantities of a phenolic colored bluish green. These reactions show t h a t proto- substance had also distilled over, as part of the silver catechuic acid was formed, and not gallic acid. This nitrate added was reduced t o metallic silver. The was further confirmed by the fact t h a t the character- solution was heated, the metallic silver filtered off, istic reaction of gallic acid with lime water, white pre- and upon cooling, the silver salt crystallized out in cipitate quickly changing t o blue, could not be ob- the form of the characteristic nacreous laminae of tained. Instead, lime water produced a beautiful silver acetate. This salt was found t o contain 65.10 purple color, which was, however, not due t o pyrogallol, per cent of silver, against 64.63 per cent calculated. because it was quite permanent, while the similar It was, therefore, free from propionic acid or other color produced by pyrogallol quickly changes t o brown. homologues. The results obtained show t h a t the tannin of the We are unable t o say what this substance, produced in addition t o protocatechuic acid, consisted of. The sugar cane is a pyrocatechin, derivative, closely remelting point of the protocatechuic acid was 183 O , sembling the oak tannins investigated by Trimble.’ against 199’ for t h e pure substance. A larger and However, it is not precipitated by Stiasny’s reagent,2 purer sample of this acid was produced in the potash formaldehyde and hydrochloric acid. fusion described below. SUMMARY The acid solution remaining after treatment of the The polyphenol of t h e sugar cane giving a green tannin with hydrochloric acid and after extraction with color with ferric salts is not pyrocatechin. I t is a ether was neutralized with sodium carbonate, and true tannin, giving a precipitate with gelatin, and is, purified by precipitation with lead subacetate. The like the oak tannins, derived from pyrocatechin, not lead in the filtrate was removed with sulfuric acid, the from pyrogallol. H e a t alone produces pyrocatechin, excess acid neutralized with sodium carbonate, and the and no pyrogallol; dilute acids give .rise to a phloresulting liquid tested with Fehling’s solution. A baphene and protocatechuic acid, but not ellagic or precipitate of cuprous oxide was obtained which would gallic acids; potash fusiOn yields protocatechuic and point to the glucoside nature of the tannin. But in acetic acids, but no gallic acid or phloroglucin. the author’s opinion this is no positive proof, because LOUISIANA SUGAREXPERIMENT STATION glucose may be present in the form of an adsorption AUDUBONP A R K , N E W ORLEANS, LOUISIANA compound, and the glucoside nature of the tannin could only be definitely proven if t h e tannin were crystalline LOW TEMPERATURE-VACUUM FOOD DEHYDRATION and contained glucose in definite proportions. By K . GEORGE P A L K , EDWARD M . FRANKEL AND RALPH H. M C K E E ACTIOX O F PLxALI-This was tested by first boiling Received June 18, 1919 5 g. of the tannin for 2’/2 hrs. with I O O cc. potassium INTRODUCTION hydroxide solution, 1.2 sp. gr. The product obtained did not give any reaction for caffeic acid, with ferric I n connection with the work of the Division of Food salts or phloroglucin-hydrochloric acid. The alkaline a n d Nutrition, Medical Department, U. S. Army, solution was evaporated t o dryness in a copper beaker Major (later Lieutenant Colonel) John R. Murlin and then heated t o fusion. The resulting mass was 1 “The Tannins,’’ 2, p. 90. 9 Chem. Zcntr., 2 (1908). 1832. dissolved in water, the solution acidified with dilute
