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Vol. 13, No. 6
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
(16) Ibid., p. 990. (17) Moerck, F. X., and Hughes, E. J.. A m . J . Pharm., 94, 65&5 (1922). (18) Nylen, P., 2. anorg. aZZgem. Chern., 229, 30-5 (1936). (19) Pascal, P., Compt. Tend., 177, 1298-1300 (1923). (20) Rudy, H., and Schloesser, H., Ber.. 73B, 484-92 (1940). (21) Salih, Mme. R., Bull. SOC. chim., [5] 3, 1391-6 (1936). (22) Sanfourche, A.. and Focet, B.. Ibid.. (41, 53, 963-9 (1933).
Simmich, H . , Angew. Chent., 48, 566 (1935). Travers, A,, and Chu, Y. K.. HeZv. Chim. Acta, 16, 913-17 (1933:. Treadwell, W. D., and Leutnyler, F., Ibid., 20, 931-6 (1937). I b i d . , 21, 1450-9 (1938) Wilkie. J. M., J. SOC.Chem. I n d . , 28, 68-9, 464, 980 (1909); 29, 794-9 (1910). (28) Wurzschmitt, B., and Schuhknecht, IT..Angew. Chem., 52, 711-8 (1939).
(23) (24) (25) (26) (27)
Determination of Succinic Acid in Plant Tissues G E O R G E W. P U C H E R AND H U B E R T B R A D F O R D VICKERY C o n n e c t i c u t .4gricultural E x p e r i m e n t S t a t i o n , N e w H a v e n , Conn.
Succinic acid is extracted from plant tissue with ether, freed from contamination with other substances by oxidation, converted into its anhydride, and condensed in toluene solution with p-toluidine to the insoluble crystalline succinyl-p-toluide. The properties of this substance are such as to permit of substantially quantitative isolation, and of identification by means of
S
UCCINIC acid is now known to be a metabolite involved
in the respiration system of certain animal tissues ( 1 , 2 , 8, l4,16),and current speculation suggests (4,21) that it may play an analogous part in plants as well. Accordingly, the recognition and, if possible, the accurate determination of succinic acid in plants become a matter of concern to the further development of our understanding of the physiological functions of the organic acids that form so important a part of most plant tissues. Early methods of determining succinic acid, most of which depend on the precipitation of an insoluble inorganic salt, were thoroughly reviewed in 1909 by von der Heide and Steiner (11) who recommended the use of the silver salt a5 being the most satisfactory. More recent modifications of the silver salt method, designed for application to the analysis of animal tissues, have been described by several investigators (6, 7,9,16). During the past few years, however, attention has been directed chiefly to methods that depend on the oxidation of succinic acid in the presence of an enzyme that occurs in muscle tissue (2, 12, 22). These methods are especially suited to the estimation of the minute amounts of succinic acid that are encountered in the small samples of animal tissue employed for the study of the details of metabolism. For work with plant tissues, none of these methods is completely satisfactory. Our knowledge of the qualitative composition of the mixture of organic acids present in most plants is very limited and salt precipitation methods are specific only under the most carefully controlled conditions. The enzyme method, although probably specific, requires the use of the IF'arburg manometric apparatus and of a highly specialized technique, and its applicability to the conditions encountered in the plant field is still to be established. It seemed desirable, therefore, to develop a simple method which would permit the isolation of a characteristic derivative of succinic acid in order to provide both qualitative and quantitative evidence of the tissue composition.
the melting point and crystalline form. An empirical solubility correction and a conversion factor are provided that lead to average recoveries of the order of 99 per cent, and single determinations can be made within * 5 per cent over the range from 1 to 20 mg. of succinic acid. The only known interfering substance is a-ketoglutaric acid.
Auwers, in 1896 (S), observed that anhydrides of organic acids condense with certain aromatic amines to give insoluble crystalline derivatives. Succinic acid is readily converted into its anhydride when heated with acetyl chloride (6),and the condensation product of succinic anhydride with p-toluidine mentioned by Auwers is a well-crystallized substance that is almost insoluble in toluene and possesses excellent properties for quantitative isolation and for identification. The reactions involved are: CHZ-COOH I CHZ-COOH
CH3COCl-
Succinic acid Molecular weight
CH2-CO
1
CHpCO/
' 0
+ H*N.C$Hd.CHI p-Toluidine
Succinic anhydride
118.1
CHz-CO-NH-CeH4.CHa
--+ AH2-COOH Succinyl-p-toluide Molecular weight 207.2 The theoretical yield from 1 mg. of succinic acid is 1.75 mg. of toluide. Although the use of amines of even larger molecular weight would appear to be desirable, none of those tested gave condensation products with as favorable properties as the p-toluide.
Preparation of Organic Acid Fraction Fresh plant tissue is prepared for analysis by being dried in it ventilated oven at 80' C. and is then ground t o a powder. Of this, 1.0 gram is accurately weighed and is extracted with ether its described by Pucher, Wakeman, and Vickery (17). The ether extract is treated with 25 ml. of water and the ether is evaporated. Troublesome frothin is sometimes encountered if alkali is added before evaporation. %he solution is diluted with 25 ml. of water,
June 1.5, 1941
ANALYTICAL EDITION
'2 ml. of 5 N sodium hydroxide are added, and the flask is agitated
until all residue is dissolved. The solution is acidified by the addition of 3.5 ml. of 18 N sulfuric acid; 5 ml. of cold saturated barium hydroxide solution (to provide a solid phase to assist in the removal of undesirable contaminants) and 0.5 gram of asbestos are added and the mixture is boiled gently for a few minutes, cooled, and filtered with suction through a layer of asbestos into a 150-ml. beaker marked a t a volume of 75 ml. ( A convenient apparatus for this and subsequent filtrations is constructed from a small bell jar with side tubulature for the vacuum line and top tubulature for the introduction of the funnel that supports the Gooch crucible. The bell jar rests on a lubricated glass plate on which the beaker is placed on a small block of wood with a hole to accommodnte it. A small desiccator can be substituted for the bell jar.) The flask and asbestos are washed repeatedly with small amounts of water until a total volume of 75 ml. of filtrate is reached. The solution is then heated for 10 minutes in a boiling water bath, 30 to 35 ml. of 1.5 N potassium permanganate are added, and heating is continued for about half an hour. From time to time the solution is stirred and examined (spot test on filter paper) for the presence of excess permanganate, more being added if necessary. An excess must still be present a t the end of the oxidation. The hot solution is then treated with a freshly prepared 20 per cent solution of anhydrous sodium sulfite until decolorized and 0.5 ml. excess is added. After being evaporated to about 25 ml. on the steam bath, it is filtered into the tube of a continuous liquid extraction apparatus and is extracted with alcohol-free ether overnight. A suitable apparatus, modified from that of Handorf (IO), which is placed in an ordinary Soxhlet apparatus has been described by Vickery and Pucher (20),and a somewhat similar device is described by Quick (18). The ether extract is treated with 5 ml. of water and most of the ether is evaporated in a water bath, the aqueous residue being finally boiled for a few minutes. It is then chilled and washed into an Erlenmeyer flask fitted with a standard taper joint for the subsequent attachment of a reflux condenser, and is evaporated, finally on a steam bath, to dryness. The evaporation of the succinic acid solution must be conducted with great care t o avoid loss by volatilization. A hot plate may be used in the early stages, but not at the end. I n order to ensure the com lete oxidation of all organic substances except succinic acid, t i e dry residue is treated with 0.2 i d . of concentrated hydrochloric acid and 0.1 ml. of concentrated nitric acid and is again evaporated to dryness on the steam bath. (Tests with 2.5 times these quantities of nitric and hydrochloric acid showed that no detectable loss of succinic acid occurs.) The flask is then cooled and is placed in a vacuum desiccator charged with sulfuric acid and provided with a small container of solid sodium hydroxide, and is left under vacuum until all traces of volatile acid have been removed; this usually requires 4 to 5 hours, but it is best to allow the sample to remain overnight.
Dehydration of Succinic -4cid The dry residue of succinic acid is treated with about 0.5 ml. of redistilled acetyl chloride (boiling point, 50' to 54" C.) and is heated for an hour with occasional shaking under reflux in an all-glass apparatus in an oil bath a t 55' to 60" C. Protection from moisture is desirable. The flask is then raised out of the oil bath and allowed to cool for 3 or 4 minutes and is placed in a desiccator that contains sulfuric acid and a container of solid sodium hydroxide, and allowed to stand overnight a t room temperature. The desiccator milst not be evacuated. Losses of succinic anhydride have invariably been encountered if attempts are made to hasten the rate of evaporation of the acetyl chloride by the use of heat or vacuum and this step has been found to be one of the most critical for the success of the analysis.
Formation of Succinyl-p-Toluide Five milliliters of boiling toluene are added to the flask, which IS then covered with a glass bulb or watch glass, and is heated to maintain boiling for about 5 minutes; 50 mg. of tricalcium phosphate are added as filter-aid and the solution is filtered with gentle suction into a 10-ml. beaker through a 1.5 ml. dry Gooch crucible provided with a thin mat of asbestos. The flask is washed twice with 1 ml. of boiling toluene and the crucible is finally rinsed with 0.5 ml. of the same solvent. The clear filtrate is treated with 0.5 ml. of a 6 per cent solution of p-toluidine in toluene (reagent reserved in the refrigerator) and the mixture is stirred with a i n e rod. This is removed, rinsed with a few drops of toluene, and t,he covered beaker is allowed to stand a t room temperature for 5 minutes and is then warmed in an oil bath a t 60" to 65" C. for 10 minutes. The beaker is then removed from the bath, allowed
413
to cool to room temperature for 10 minutes, chilled in a bath of water a t 4" to 5' C., and placed in the refrigerator for an hour. After about 15 minutes, a rod is introduced and the crop of crystals is occasionally stirred. Filtration is accomplished on a 1.5-ml. dry, weighed Gooch crucible furnished with a thin asbestos pad. The filtrate is collected in a graduated cylinder to provide a check on the volume and is used as necessary, together with a rubber policeman, to complete the transfer of the crystals to the filter. It is kept cold during the filtration and other manipulations by being immersed in a beaker of ice water. The precipitate is finally sucked dry, washed twice with 1 ml. of a saturated solution of succinyl-ptoluide in toluene, dried a t 105" C. for half an hour, and weighed. The wash fluid employed is prepared from a sample of pure succinyl-p-toluide made by treating 0.5 gram of recrystallized succinic anhydride (19) with 0.6 gram of p-toluidine in 15 ml. of chloroform. The mixture is warmed to the boiling point for 30 minutes and is then cooled to 5' C. The crystals are filtered and washed with cold chloroform and should melt a t 178" C. uncorrected. Of this material, 50 mg. are heated with 500 ml. of toluene for a short time and then chilled in the refrigerator overnight; after being filtered the solution is kept at low temperature.
Calculation of Results Succinyl-p-toluide is not entirely insoluble in toluene undei the conditions employed nor is the conversion of succinic acid into this product quantitative. However, the conditions have been adjusted so a s t o admit of repetition in exact detail and an empirical solubility correction and a factor to convert the results into substantially accurate determinations of succinic acid have been established. T h e weight in milligrams of t h e succinyl-p-toluide derived from 1 gram of tissue, when inserted into the following formula, yields the percentage of succinic acid. (Weight of succinic-p-toluide 0.34) 0.059 = per cent succinic acid. The d a t a upon which this formula rests are discussed below.
+
Identification of Succinic-p-Toluide Although when prepared from pure succinic acid, this product is colorless and well crystallized, samples obtained from plant tissues are occasionally dark i n color and may not be entirely typical in appearance. Proof of identity is therefore desirable, a s well as some index of purity. The melting point has been found to provide this information and, together with the appearance under the microscope, serves for complete identification. The quantity of material available is frequently so small, however, that special technique is required for the preparation of crystals of typical appearance.
A convenient method is to treat the dry product in the crucible with 1 ml. of acetone, which dissolves it readily, and draw the solution gently through into a 5-ml. beaker placed beneath it in the filtration apparatus. The asbestos is stirred with a fine rod and transfer is completed with a second equal quantity of acetone. Suction is maintained until the whole of the acetone has evaporated, and the beaker and crucible are then dried a t 105" C. for a few minutes to remove the last traces of acetone. One milliliter of methylamyl acetate (methylisobutylcarbinol acetate) is added to the beaker, which is then warmed on a hot plate until the residue is dissolved (1 ml. of boiling methylaniyl acetate will dissolve about 10 mg. of succinyl-p-toluide), 1 ml. of boiling toluene ia added, and the mixture is filtered through the original crucible into a 10-ml. beaker. The original beaker is rinsed 3 times with 2-ml. portions of hot toluene which are also drawn through the filter. The filtrate is heated in case crystallization has begun, and is then chilled in the refrigerator and, when cool, is seeded with a tiny crystal of pure succinyl-p-toluide and stirred until crystallization be ins. After being allowed to stand overnight, the crystals are htered on a small Hirsch funnel on hardened filter paper, washed with cold toluene, and dried a t 105" C. A sample of the product is mounted in water for examination under the microscope and the melting point is determined.
A product t h a t melts completely t o oil at or above 173' C. uncorrect,ed, as measured with a long-stem thermometer. is
INDUSTRIAL AND ENGINEERING CHEMISTRY
414
Vol. 13, No. 6
Justification for the use of this correction is shown in Table TABLE I. YIELD OF SUCCINYL-p-TOLUIDE FROM SUCCINIC ACID Succinic Acid Taken
Toluide Found
Mo.
Mg.
1.00
1.33 1.29 1.31 1.34 1.73 1.72 1.77 1.77
1.25
5.00
Mean Ratio of Toluide to Acid (Theory I . 75)
1.32
1.40
5.21 8.04 5.34 5.25
1.64
1.70
regarded as being satisfactorily pure. I n general the melting point lies between 175" and 177" C.; the pure substance melts at 177"to 178" C. The crystals are long narrow parallel-sided prisms or needles, the ends of which are usually square. Occasionally somewhat stout rectangular] but often broken, prisms are formed. Comparison with authentic pure material crystallized from the same solvent must be made until familiarity with the appearance of the substance is acquired. When only about 2 mg. of the product are available, recrystallization may be effected with 5 d.of boiling toluene alone and the filtration omitted. Under these circumstances typical crystals are less easily secured.
TABLE 11. RECOVERY OF SUCCINIC ACID AS SUCCINYLp-TOLUIDE Succinic Acid Taken
Toluide Found
Toluide Calculated
M9.
M Q.
M Q.
1.00 1.25 2.50 5.00 10.0 20.0
1.34 1.75 3.80 8.21 16.95 33.88
1.75 2.19 4.39 8.77 17.54 35.08
Succinic Acid Recovered CorCorrected rected for soluUnfor bility corsohand by rected bility factor
% 76.4 79.8 86.7 93.6 96.6 96.6
%
%
95.8 95.3 94.4 97.5 98.5 97.6
99.1 98.7 97.7 100.9 102 0 100.9
I1 which gives the results of a series of determinations, each figure being the mean of four or more separate values. Addition of the solubility correction brings all the results obtained over the range 1 to 20 mg. of succinic acid to essentially the same level of recovery. The maximum discrepancy even for the smallest quantities is only 5 per cent. The mean of all is 96.5 f: 1.6 per cent. To allow for losses over the entire procedure, including failure to accomplish complete conversion of the acid to its anhydride and to bring about complete condensation to the toluide, an empirical factor is used to convert the weight of the toluide to succinic acid. The theoretical value of this factor is 0.570; if this is increased to 0.590-that is, by 3.5 per cent-the recoveries shown in the last column of the table are secured. The mean of these is 99.9 * 1.6 per cent over the entire range of quantities considered.
Succinic Acid in Plant Tissues A series of determinations of succinic acid in several tissues is shown in Table 111. This acid is a minor constituent as compared with the malic or citric acid such tissues as these usually contain, and its significance as a metabolite still awaits full explanation. From the point of view of the present paper, it is necessary only to establish the validity of the results as determinations of succinic acid. Aside from the reproducibility, which is satisfactory, the most useful criteria are the melting point, the crystalline form, and the color of the product. The melting point of material derived from pure succinic acid is 177" to 178" C. uncorrected. However, when working with small quantities under the conditions described, melting points as low as 173" C. are frequently encountered, and the preparations are not always white after being dried. Moreover, the melting points of the products in the replicate tissue analyses in Table I11 vary within these limits and there are differences in the color, although there is no serious variation in the yields.
TABLE 111.
Table I shows the results of analyses in which from 1.0 to 20.0 mg. of succinic acid were taken. Aliquot parts of a standard solution were evaporated to dryness in the flasks and were then treated as described. The replications show that consistent results are readily secured, but the recoveries of the smaller amounts of acid are low. Clearly the solubility of the toluide, under the conditions of the analysis, cannot be neglected. Solubility determinations, made by evaporating the saturated solution of the toluide in toluene and by calculation from recoveries of toluide derived from weighed amounts of succinic anhydride, led to a mean value of 0.34 mg. in 7 ml. of toluene a t the working temperature of 5" to 9" C. This figure is empirical, since the values calculated from re covery experiments contain the errors due to losses in the manipulations. It of course applies only to analyses conducted exactly as described. The observed solubility in 7 ml. of toluene was close to 0.30 mg.
Trssms
ACID IN LEAF
Melting Point (Uncorr.)
Wt. of Sample Gram8
Wt. ,of Toluide
Mg.
%
OC.
Tobacco
1.0 1.0 0.5 0.5
9.03 9.24 4.09 4.21
0.55 0.56 0.52 0.53
173 177 175 173
White Pale brown Brown White
Bryophyllum
0.5 1.0
1.39 2.91
0.19 0.20
173 176
Brown White
Maize
1.0 1.0
3.27 2.74
0.21
174 173
White Pale brown
t
Recovery of Pure Succinic Acid
DETERMINATIONS O F SUCCINIC
Succinic Acid
0.18
Color of Toluide
TABLE IV. RECOVERY OF ADDEDSUCCINIC ACID Succinic Acid Present 50 mg. of citric
+ 100
mg. of malic acid
% 0.0 0.0
Succinic Acid Added
MQ. 10.0 10.0
Recovery
% 99.6 103.5
Tobacco leaf
Rhubarb leaf
0.57 0.50 0.50
5.0 2.5 2.5
0.03 0.03
5.0 6.0
99.8 104.0
ANALYTICAL EDITION
June 15, 1941
That a contamination of the toluide that depresses the melting point to 173” C. is without significance as regards the validity of the analytical data is apparent from the results of a test in which succinic acid was added to a sample of rhubarb leaf blade tissue which yielded only 0.17 mg. of apparent toluide per gram; the succinic acid content was thus of the order 0.03 per cent or less. Five milligrams of succinic acid were added to 1 gram of this and recovered, The resulting toluide weighed 8.30 mg., the equivalent of 5.1 mg. of succinic acid, and melted at 173’ C. If it be assumed that the whole of the product derived from the tissue itself represents a contamination, the recovered toluide would have contained 2.5 per cent of impurity, a quantity that would not significantly affect the accuracy of an analysis by the present method, and its melb ing point fell within the limits found when dealing with recoveries of succinic acid taken as such rather than added to tissue. Additional individual data for the recovery of succinic acid from a mixture with other acids and from tissues of different succinic acid content are shown in Table IV; all save one are within * 5 per cent.
Discussion Comment on the steps of the procedure is perhaps superfluous, save to say that the technique of working with small amounts of material must be acquired by patient practice. The conditions under which the toluide is crystallized have been described in great detail, since the appearance of the final product is a matter of concern in the identification, and the validity of the calculation of the results rests upon adherence to the directions. Success is dependent on the complete removal of acid and of acetyl chloride before the condensation is attempted; if the product separates a t once when the toluidine is added, this has not been accomplished and the analytical results are worthless. Precipitation a t this point should not begin for several minutes, depending on the amount of succinic anhydride present. However, no tendency towards supersaturation has been noted when the solution in toluene is chilled; the product at this stage may be crystallized or may separate in a granular form. The certain production of typical crystals requires the recrystallization step. Succinyl-p-toluide is too soluble in ether, chloroform, or other common solvents to permit of convenient recrystallization of small amounts. It dissolves in hot methylamyl acetate easily but separates sluggishly when the solution is cooled; accordingly the filtration is readily accomplished and, by means of the addition of toluene, a fairly complete final separation is brought about after the solution is seeded and chilled. The loss is small and sufficient material for a melting point can be obtained from as little as 1mg. under the conditions described. It is important to note that these conditions are selected t o bring about only a minimum purification, so that the melting point of the recrystallized product may as truly as possible indicate the purity of the material weighed. The specificity of the method depends upon the success with which the toluene solution has been freed from substances, other than succinic anhydride, that condense with toluidine. The original extraction of the organic acids from the tissue provides for the separation from carbohydrates and amino acids-. g., glutamic acid- that might yield succinic acid on oxidation. Other organic acids are destroyed by the oxidation with permanganate and the remaining traces of interfering substances in the second ether extract are destroyed by aqua regia. Omission of this step frequently leads to the formation of an oily condensation product. The most critical points of the procedure are the evaporations, particularly that of the excess of acetyl chloride. For
415
this reason redistilled pure reagent must be employed and access of moisture should be prevented but, owing to the volatility of the anhydride, the use of elevated temperature or vacuum is inadmissible. The preparation of the plant tissue for the analysis requires no especial remark save that, in the case of material such as seed tissue that contains true fats, previous extraction with ether to remove the fat should not be attempted because of the danger of loss of succinic acid. Fat itself does not interfere, since it is removed during the preparation of the solution for oxidation. A test of tobacco-seed oil subjected to the whole procedure gave a hardly weighable trace of condensation product. However, if removal of fat previous to the succinic acid determination is necessary, the extraction should be done with petroleum ether. No loss of succinic acid is to be anticipated if this solvent is used. The only substance present in plant tissues that would be expected to interfere with the present method is a-ketoglutaric acid. This substance is quantitatively oxidized to succinic acid by permanganate (IS) and its properties are such that it would follow the succinic acid to the oxidation step. Accordingly the results of the present method as applied to plant tissues include a-ketoglutaric acid. However, only traces of this substance have been reported in plants in the few cases where it has been identified a t all, and in any case, according to present theory, i t is closely related in metabolism to succinic acid. Attempts to provide a t least qualitative tests from which some estimate of the significance of the influence of this possible contaminant may be secured are now in progress. The method is designed to deal with samples of tissue not larger than 1 gram; practical difficulties are encountered if larger samples are taken. The lower limit of certain identification of succinic acid is about 1 mg. and the quantities of reagents are adjusted to care for a maximum of 25 mg. The range of the method is thus from about 0.1 to 2 per cent of succinic acid in the dry tissue; for tissues richer than this smaller samples should be taken. Between these limits a precision of * 5 per cent for a single determination should be readily attainable.
Literature Cited Annau, E., Banga, I., Blazd, A,, Bruckner, V., Laki, K., Straub, F. B , and Szent-Gyorgyi, A., 2. phy8iOl. Chem., 244, 105 (1936).
Annau, E., Banga, I., Gozsy, B., Huszak, St., Laki, K., Straub, B., and Szent-Gyorgyi, A., Ibid., 2 3 6 , l (1935). Auwers, K., Ann., 292, 132 (1896). Chibnall, A. C., “Protein Metabolism In the Plant”, New Haven, Yale University Press, 1939. Clutterbuck, P. W., Biochem. J.,22, 745 (1928). Fieser, L. F., and Martin, E. L., Org. Syntheses, 15, 93 (1935). Goepfert, G. J., Biochem. J., 34, 1012 (1940). Gozsy, B., and Szent-Gyorgyi, A,, 2. physiol. Chem.. 224, 1 (1934).
Hahn, A., and Haarmann, W. Z., 2. Biol., 89,159 (1929). Handorf, H., 2. angew. Chem., 35, 257 (1922). Heide, C. von der, and Steiner, H., 2. Untersuch. N a h r . u. Genussm., 17, 291 (1909).
Krebs, H. A,, Biochem. J . , 31, 2095 (1937). Ibid., 32, 108 (1938). Krebs, H. A., and Eggleston, L. V., Ibid., 34, 442 (1940). Krebs, H. A., and Johnson, W. A., Enzymologia, 4, 148 (1937). Moyle, D. M., Biochem. J., 18, 351 (1924). Pucher, G. W., Wakeman, A. J., and Vickery H. B., IND.ENG. CHEM, Anal. Ed., 13, 244 (1941). Quick, A. J., Ibid., 5 , 76 (1933). Shriner, R. L.. and Struck, H. C., Org. Syntheses, 12, 66 (1932). Vickery, H. B., and Pucher, G. W., Conn. Agr. Expt. Sta., B u l l . 323, 197 (1931). Vickery, H. B., Pucher, G. W., Wakeman, A. J.. and Leavenworth, C. s.,Ibid., B u l l . 424 (1939). Weil-Malherbe, H.. Biochem. J.. 31, 299 (1937).
