Alanine in Rubber Latex J . MCGAVACK AKD J. S. RUMBOLD, United States Rubber Company, Passaic,
IV. J.
i:yp
N D I R E C T evidence of the Serum j r o m normal latex obtained by creaming ~ ~~ u ~~ ~ ~ f ~ presence of amino acids in in the usual manner is dried to a thick sirup. vlscous sirup, 200 cc of absolute This is dissolped in absolute alcohol and amino a l c o h o l \%erea d d e d , a n d t h e o r d i n a r y ammonia-preamino acids were ePterified by served latex was Obtained from acids esteriJied in the presence of dry hydrochloric saturating the alcohol Trith dry electrometric titration work with acid. B y fractionaf ion and extraction methods, the glass electrode, the applicaThe alcohol n as gas evaporated off zn vacuo belo\\ 50" C., and t h e tion of mhich t o latex was decrystalline material with a sharp melting point scribed in a previous article ( 2 ) . having 15.5 per cent nitrogen is obtained. T h i s ~ ~ P , o ~ ~ ~ d a A small sample of has is identified as d-alanine by electrometric titraThe nearly black viscous residue obtained by evaporation of the alnow been isolated and identition using the glass electrode. The indications cohol was dissolved in one-third fietl, a n d i n d i c a t i o n of t h e are that /atex contains about 0.03 per cent of this to one-half the volume of water presence of phenylalanine has and Doured into a 2-liter flask. a l s o b e e n obtained; however, substance. Seven hundred cubic centimeowing to the small amount of ters of ether were added, and the mixture was cooled in a freezing mixture. Ice-cold 30 per cent material, its identification could not be established. hydroxide was added in small quantities a t a time until It is believed that this is the first amino acid t o be isolated sodium the hydrochloric acid was neutralized, and then solid potassium from latex in the pure state and positively identified; the carbonate; the flask was continually shaken in the freezing fact that it has not been detected before may be due t o the mixt,ure. The ether was poured off and replenished by a fresh small amount usually present and t o the great solubility of quantity. Solid potassium carbonate and sodium hydroxide soluwere added gradually t'o form finally a thick mass which was the substance. The only reference found in the literature tion shaken out repeatedly with 500-cc. lots of ether until the ether relating to the presence of a n amino acid in rubber is the separat,ed in a nearly colorless state. About 4 quarts (4.6liters) article by Whitby, Dolid, and Yorston ( 3 ) . These workers of ether were required for the extract,ion. The ethereal extracts were dried by shaking for about 10 obtained a small amount of d-valine from the acetone extract minutes wit,h potassium carbonate, and were then combined and of pale crepe. lett overnight with anhydrous sodium sulfate. Glycine, proline, and leucine are apparently either absent The ether was evaporat>edin small quantities at a time by or present in relatively small amounts. If the amino acids passing a current of dry air over the solution at the ordinary are derived from decomposition of proteins, the absence of temperature. A light brown oil was left which was fractionally in vacuo, using a Cenco vacuum pump, the receiver glycine is rather rernarkable since most proteins with a high distilled being cooled with solid carbon dioxide. The following fractions alanine content-for example, silk fibroin-also contain this were collected : amino acid. Allowing for unavoidable loss, the amount of alanine isoFRACTION TEMP. PRESSURE WEIQHT c. -1.im . Grams lated in the pure state is, as was expected, not enough t o I 20 11.0 41.8 account for slope at the half-way point of the latex potentioI1 20- 60 11.0 12.1 metric titration curve. Although reasonable precautions I11 6&100 5.0 2.0 Rmidue . . . . .. 2.0 were taken to avoid loss, no effort was made to obtain quantitative data, but from the amount of pure alanine obtained ISOLATIOS OF ~-ALSKIKE (about 1.3grams from i liters of latex serum) it iq thought that the alanine content of the original latex could not have been The first fraction consisted almost entirely, and the more than about 0.03 per cent. A solution of alanine of this second very largely, of alcohol which had been dissolved by strength is about 0.003 N. It appears likely that there the ether, since the viscous nature of the original sirup premust be several other amino acids of which the dibasic vented complete removal of the alcohol used in the esterificaglutamic acid, commonly occurring in plant proteins, may tion process. be one. Fractions I1 and I11 were colorless oily liquids, which,
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METHOD OF EXTRACTION The method employed for the extraction of the amino acids was the classical one of Emil Fischer. Chiefly on account of the presence of quebrachitol and a large amount of soluble impurities, attempts to obtain an amino acid b y the action of solvents on the dried serum always failed. The only substance obtained b y this method mas quebrachitol, which crystallized readily from alcohol after repeatedly boiling the solution with animal charcoal. The following procedure was successful in isolating an amino acid: About 7 liters of latex serum, prepared by creaming some ammonia latex with ammonium alginate, were stirred in a current of air until there n a 5 no odor of ammonia. The object of this preliminary treatment mas to reduce the amount of ammonium chloride n hich mould be formed on addition of hydrochloric acid, but it was prohablv unnecessary. The residual rubber, etc., was coagulated by a little dilute hydrochloric acid and removed by filtration. The resulting
when added to water, dissolved completely to give strongly alkaline solutions. (The amino acid esters are strong bases owing to the blocking of the carboxyl group by the ethyl radical.) The solutions were boiled with water under a reflux condenser until the alkaline reaction disappeared and were then evaporated to dryness in weighed flasks. The total weight of residue from fractions I and I1 was only about 2.5 grams. The constituents might be glycine, alanine, leucine, and proline. The residues were combined and extracted several times with boiling absolute alcohol, and the alcoholic extracts were evaporated to dryness. This treatment removed some of the coloring matter but apparently very little else, and it may therefore be concluded that proline, which is appreciably soluble in alcohol, was either absent or present only in very small amount. The amino acids insoluble in alcohol were dissolved in water and completely decolorized by boiling the solution with
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INDUSTRIAL AND ENGINEERING CHEMISTRY
animal charcoal; the solution was then slowly evaporated on a water bath. However, it had to be taken down to very small b u k before any crystals appeared. They were filtered off, alcohol was added to the filtrate until the mixture became slightly turbid, and it was heated on the water bath for a few minutes and cooled in ice. A good crop of small colorless needles was obtained; they melted at 286' C. (uncorrected) and had a sweet taste (melting point of d-alanine, 297" C.). Three recrystallizations from aqueous alcohol raised the melting point to 290" C. (corrected), and after seven recrystallizations the compound melted at 295 o (corrected). since the first crop of crystals apparently also consisted chiefly of the same substance, it was purified in the same way and combined with the rest. The crystals melted in a capillary tube to a brown liquid, which decomposed with a rapid evolution of gas. A sample of d-alanine (melting point, 293" C.) had a melting point determined by the same thermometer of 294". Further characterization was obtained by a determination of the nitrogen content: found, 15.5 per cent; calculated (for alanine), 15.7. The residue from fraction I11 was too small to be further examined.
EXAMINATION OF RESIDUE(UNDISTILLED, HIGHERBOILING ESTERS) The brown sticky substance left in the distilling flask was extracted with ether, and the ether was evaporated in uacuo. The resulting dark brown oil was mixed with about 5 volumes of water; only a part dissolved, the rest remaining as a brown oil. This indicates that a considerable amount of the ethersoluble portion of the residue consisted of phenylalanine ester, which differs from the esters of aspartic acid, glutamic acid, and serine by being only slightly soluble in water. The aqueous solution containing the insoluble oil was extracted with an equal volume of ether, and the ether extract was washed several times with water to remove traces of any of the other esters which might have been dissolved. The ether was removed, and the ester hydrolyzed by boiling with concentrated hydrochloric acid. It gave a dark brown solution. Crystals could be seen on the side of the flask, and a brown insoluble substance was formed. The solution was boiled with animal charcoal, filtered, and evaporated to dryness. A crystalline substance was left, but it was too small to be identified.
Vol. 26, No. 7
removing 10-cc. samples and titrating them with standard hydrochloric acid, and the pH by means of a glass electrode. The results are plotted in Figure 1. The true titration curve obtained by methods well recognized shows that the end point is a t 0.0244 N . Here the concentration of organic material was : 'Oo0
0.5436
1001.3
grams per liter
Hence the molecular weight of the compound is: 1000 X 0.5436 X 4 = 89.0 1001.3 X 0.0244
l o l l 9
!
E N D ;POINT 41
sz
NaOH NORMALITY
.03
.04
FIGURE1. TITRATION CURVEOF LATEXAMINO ACID AT 25' C.
This value, which can be taken as the molecular weight rather than merely the combining weight (since, under the conditions of the vacuum distillation, only monobasic, amino acid esters could have distilled) agrees with alanine (molecular weight 89.1). A rough estimation of the molecular or combining weight can be obtained from the slope of the curve a t the half-titration point by means of the approximate formula : d [ B 1 = 0.5758 [SI d(pH) Where B = equivalents of NaOH added S = total amino acid in all forms Square brackets refer to equivalent concentration
The reciprocal of slope a t the half-titration point is 0.0146. Hence, molarity = 0.0146/0.5758 = 0.0254, and the a p By means of the titration curve we can determine the acidic proximate molecular weight = (0.5436 X 4)/0.0254 = 86. At the half-titration point the pH is 9.71. This gives dissociation constant and the combining weight of the amino K. (25' C.) = 1.95 X 10-lo. The same electrode gave the acid. same value for Ka for a sample of d-alanine (data not given). TITRATION OF A SOLOTION OF LATEX The value of K , (25" C.) for alanine reported by Branch and TABLE I. ELECTROMETRIC AMINOACID" WITH A & , A s s ELECTRODE Miyamoto (1) is 2.06 X 10-lo. PH NORThe long method reported here for the extraction of an FROM MALITY OF NORPOTENTIOMETEB CALI- NaOH IN MALITY OF CORRECTED amino acid from latex is probably not the simplest that could READINQ AT ELECTRO-BBAAMINO .4QUEOUS NORbe devised; it was adopted chiefly because it seemed the one Null 0.5810 MOTIVE TION ACID NnOH AT MALITY point volt FORCE C U R V B SOLN. SAME PH OF NaOH most likely to give definite results, 517.5 725 0.4147 8.83 0.0028 . . .. 0.0028 It would be useful to know whether the amino acids are 555 725 0.4448 9.23 0.0058 .. . . 0.0056 576 725 0.4616 9.44 0.0082 ,... 0,0082 present in the latex immediately after its removal from the 595 722 0.4788 9.68 0.0119 .... 0.0119 621 722 0.4997 9.99 0.0158 .. . . 0.0158 tree or are produced by alkaline hydrolysis of proteins. 656 722 0.5279 10.43 0.0197 0.0003 0.0194 In this connection it is perhaps worthy of note that, although 705 722 0.5673 11.09 0.0238 0.0013 0.0225 730 722 0.5874 11.40 0,0272 0.0028 0,0244 the available evidence is in favor of the view that latex con756 722 0.6084 11.89 0.0326 0.0082 0.0244 768 722 0.6180 12.23 0.0448 0.0180 0.0268 tains several amino acids, so far only alanine has been isolated 0.5436 mum per 250 ca. in sufficient quantity to obtain definite identification. Evidence of the presence of others is meager, and it is possible that The titration (data given in Table I) was carried out-with d-alanine is a natural constituent of latex and that the pro50 per cent carbonate-free sodium hydroxide. The concen- teins are not hydrolyzed to any great extent. Work in Sumatra, Dutch East Indies, by one of the writers tration of sodium hydroxide after each addition was found by
ELECTROMETRIC TITRATION OF THE LATEXAmxo ACID
July, 1934
I N D U S T R I A 1, A N D E N G I N E E R I N G C H E M I S T R Y
resulted in the extraction of a small quantity of a similar amino acid from a Obtained by Of dried Serum spraying the Serum left from acetic acid coagulation of Some fresh unpreserved latex. Owing to the smaller amount it could not be purified to the same extent, and its melting point was about 20" C. lower than that of the substance obtained here* The rotation in acid Of this impure product was found to be a 3 = ~ 4-6.4'Emil Fischer gives a ' : = t 1 0 . 3 for pure d-alanine hydrochloride.
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LITERATURE CITED (1) Branch and Miyamoto, J. Am. Chem. Sot,, 52, 863 (1930). (2) McGavack, J., and Rumbold, J. S., IND. ENG.CHEM:., Anal. Ed., 3, 94 (1931). (3) Whitby, G. S., Dolid, J., and Yorston, F. H., J . Chem. Soc., 1926, 1448. RECEIVED April 4, 1934. Presented before the Division of Rubber Chemistry a t the 87th Meeting of the American Chemical Society, St. Peterrburg, Fla., March 25 t o 30, 1934.
Comparative Toxicity of Fluorine Compounds MARGARET CAXMACK SMITHAND RUTHM. LEVERTON, University of Arizona, Tucson, Ariz.
R
EPORTS in the literature
they often have to be replaced The use of drinking water containing fluorine by false teeth at an early age. of t h e toxic effects of in concentrations of one part per million or Fluorine compounds are befluorides have been remore is recognized as the cause of mottled enamel, coming more and more comviewed by McClure and Mitchell a defect of human teeth (12, 13, 15). The ( 7 ) and DeEds (4). Such indimonly used as spray insecticides increasing use of fluorine compounds as spray cations of impaired health and in place of arsenicals because the development as subnormal rate toxic action of a r s e n i c a l s is insecticides has prompted this study of the of growth or decline in weight, known and f e a r e d . It would comparat ice toxicity of different compounds of appear that from the standpoint poor appetite and lowered food jluorine. The following compounds of fluorine intake, poor reproduction, and of public health, chronic intoxiare used: sodium, potassium, ammonium, inferior bone and tooth structure cation from fluorides is little to and calcium fluorides; sodium, potassium, and have been r e p o r t e d to result be desired. from relatively low intakes of S o l l m a n n , S c h e t t l e r , and barium fluosilicates; and natural cryolite (soWetzel (17) in 1921 studied the sodium fluoride. High levels of dium aluminum fluoride). A comparison is feeding have caused death. The effect of fluorine intake upon the made of their effect upon growth rate, food congrowth and food intake of albino harmful effects resulting from sumption, eficiency of utilization of food, rethe use of rock phosphates as rats. They varied the percentproduction, mortality, and teeth, when supplied m i n e r a l supplements in farm age of pure sodium fluoride in the animal r a t i o n s have been beration from 0.0002 to 0.23 per to young albino rats at the same fluorine conlieved to be due to their fluorine cent for a 1- to 4-monthperiod. centration. U'ide differences in toxicity among content (1, 5 , 10, 18, 19). A c o n c e n t r a t i o n of sodium these compounds are found when the effect upon I n 1931 proof was established fluoride greater than 10 mg. per growth, food utilization, and damage to the in this laboratory (16) that the kg. of rat per day, roughly 0.015 teeth are considered, which may or m a y not be dental defect of h u m a n teeth per cent of the ration, diminished known as mottled enamel (Figfood consumption and growth a reflection of difference in their solubility. ure 1) which is endemic in many even though the food was equally From the standpoint of initial damage to the sections of the world is caused palatable to the animals. Below teeth, hot( ever, all these compounds of fluorine by the toxic action of fluorides this level no harmful effect of are found to be equally toxic. Fourteen parts present in the drinking water the fluoride w a s n o t e d . I n per million of jluorine (from any source) in supply of the afflicted persons. these s t u d i e s , however, only An extensive survey of the congrowth and food consumption the diet of the rats leaces a mark upon the rat ditions in Arizona (13) by means were used as criteria. incisors. The significance of these Jindings in of which the concentration of In 1925 Schultz and Lamb relation to the spray residue problem and human fluorides in water supplies was (11)reported briefly that a level mottled enamel is discussed. correlated with the presence or of 0.1 per cent sodium fluoride absence of mottled enamel and in the r a t i o n interfered with its degree of severity if present, gave evidence that the use of growth rate of rats and that a n "unfavorable effect in redrinking water having a fluorine concentration above 2.7' production begins a t a level of about 0.025 per cent sodium parts per million interfered with normal tooth development. fluoride." I n the same year McCollum, Simmonds, a n d The drinking of water in the usual amount which contains as Becker (8) reported that, when sodium fluoride was added to little as 5l p. p. m. of fluorine has been found to cause a severe an adequate mixed ration a t the level of 0.05 per cent, the type of mottled enamel of the teeth, the enamel being pitted teeth of rats were observed to be abnormal in appearance a n d and corroded. Mottled teeth are not only disfiguring in defective in structure. appearance but are so defective in structure and strength that I n this laboratory (14) the feeding of sodium fluoride to rats a t 0.025, 0.05, and 0.1 per cent of the ration has in each case 1 Fluoride determination made b y the modified Fairchild ferric chloride interfered with the normal calcification of the teeth to an exmethod deacribed in Ariz. Expt. Sta. Tech. BULL 43 (1932). Unpublished data a t hand now indicate t h a t a concentration of fluorine in water of as tent varying with the Concentration. Even the lowest level little as 1 p. p . m. a8 determined by the Willard or Foster methods of analysis of feeding (0.025 per cent) resulted in defective enamel of a 8 sufficient to cause mottled enamel of human teeth. A concentration of rather severe type of the teeth of rats, dogs, and guinea pigs. 2 p. p. m. is now found to be associated with mottled enamel of the more eevere type. It has been observed also that an effect upon the teeth of the
