Micromethod for Determination of Tryptophan in Bacteria and Proteins EDWARD STEERS AND M. G. SEVAG School of Medicine, I'niversity of Pennsylvania, Philadelphici 4 , Pa. S T H E course of a study on the tryptophan nietabolism of to resistance to sulfonamides (6) the authors have used the method of Sullivan and Hess (8) with certain modifications for the determination of tryptophan. These studies led to the development of the following micromethod, which permits the analysis of samples of protein (casein, bacteria, etc.) containing as little as 6 mirrograms of tryptophan i n a IO-mI. reaction system.
1 staphylococci in relation
the maximal color development. If this heat is reduced to 20" C. by cooling, immediately after or during the addition of the acid, the color development is impaired (Table I). I n the micromethod, because of the small volume of alkali used, sufficient heat is not generated and the heat liberated in the Klett tubes is rapidly dissipated because of the larger surface per unit volume. Therefore heating this reaction mixture for 1 minute a t 56" C. enables maximum color development. Further treatment is carried out a t room temperature.
DIGESTION OF PROTEINS
Five samples of protein, casein, staphylococci (washed bacterial 9ediment dried with alcohol and ether), dialyzed samples of crystalline egg albumin, lysozyme ( I ) , and ribonuclease (J),were digested in sodium hydroxide solution in a 56" C. water bath for 1 hour. Two milliliters of 5 S sodium hydroxide were used to digest all samples except casein and tryptophan. The alkali concentration for casein and I-tryptophan (in 1% casein hydrolyzate solution) was 1 N sodium hydroxide. I t is generally recommended that tissue proteins be hvdrolyzed i i i strong alkaline solution by autoclaving for 5 hours ( 3 ) . Gunness et al. ( 4 ) recommended 10 ml. of 5 -V sodium hydroxide in sealed ampoules for autoclaving 0.5 to 1.0 gram of dry bacterial cells at 6.8 kg. (15 pounds) steam presiure (121 ' C.) for 10 hours. The authors have checked thrie procedures in the macromethod and found loss of tryptophan. The method recommended represents optimal conditions for the analysis of bacterial cells for tryptophan. The tryptophan content of the proteins studied did not suffrr destruction under these coriditions. Table 1.
Effect of Temperature on Development of Color in Determination of Tryptophan
3ubstance I-Tryptophan Casein Staphylococcal cells
Amount, Mg. 0.124 10
TemperaKlettture, Summerson ' C. Reading 45 95 45 94 200 47
Tryytophan,
7% , . .
1.23 0.61
50
66. 204 0.53 37" 75 n 21 60 48 0.13 1)uriny addition of acid heat of reaction in various systems was mainrained a t ire-bath temperature. E a r h by.etrrn was then warmed t o indicated remperatures for 15 minutes, (L
AYALYSlS
To 1 ml. of the alkaline digest of the protein containing 6 to 60 iiiicrograms of tryptophan, in a 10-ml. standard Klett-Summerson colorimetric tub; are added 0.1 ml. of Ehrlich's reagent (5% p dimethylaminobenzaldehyde in 10% sulfuric acid solution), 0.04 ml. of 2% sodium nitrate, and 5 ml. of concentrated hydrochloric acid. [This concentration of sodium nitrate represents a twofold increase over the amount recommended by Bates ( 2 ) . This amount of sodium nitrate is required for maximal color development because of volume relations and the increased amount of alkali used for digestion.] After the mixture is heated for 1 minute in a 56" C. water bath, it is allowed to stand a t room temperature for 15 minutes. The system is then diluted to the 10-ml. mark with 17.5% hydrochloric acid solution and permitted to stand for an additional 15 minutes. The sample is then read in a photoelectric colorimeter using a 560 mfi filter (Klett-Summerson So. 56). If the salt formed upon the addition of the excess acid to the alkaline solution fails to dissolve upon final dilution, the reaction system may be filtered through a dry paper before reading.
Table 11. Protective Action of Amino .4cids on Development of Color in Determination of Tryptophan KlettSummerson Reading
bystem 1. 2. 3.
124 7 tryptophan in 5 ml. Hz0 124 y tryptophan in 5 ml. H20 25 me. K a O H 124 y tryptophan in 5 ml. 1% casein hydrolyzate soln. 25 me. K a O H 10 mg. casein 4- 5 ml. Hz0 25 me. N a O H 10 mg. casein, 125 y tryptophan, in 5 mi. H20 f 25 me. NaOH
i:
+
+
+
96 51 95 94 184
The amount of tryptophan in the sample is determined by comparison of the reading with a standard curve obtained from known amounts of tryptophan analyzed in the same manner. The tryptophan standard curve is obtained by analysis of aliquots (range 6 to 60 micrograms per ml.) of a solution of 2-tryptophan in 1% casein hydrolyzate ( p H 7.35), each milliliter of which contains 60 micrograms of added tryptophan and 1 milliequivalent of sodium hydroxide. The 1 % casein hydrolyzate solution used as solvent for the &tryptophan was prepared according to the method of Straus et a2. (?), using vitamin-free 10% casein hydrolyzate manufactured by General Biochemicals, Inc. Analysis of the 1% casein hydrolyzate solution by the macro- and micromethods showed no tryptophan. When 500 ml. of the 1% casein hydrolyzate solution were concentrated in vacuo in an atmosphere of nitrogen, a t a water bath temperature of 45" C. to a final volume at 35 ml., samples were analyzed by the macro- and micromethods for tryptophan with negative results. Furthermore, the 1% casein hydrolyzate medium did not support the growth of a strain of Staphylococcus aweus which requires tryptophan for growth. The function of the 1% casein hydrolyzate solution as solvent for the I-tryptophan is to prevent any oxidation that would occur during alkaline digestion (Table 11).
Table 111. Determination of Tryptophan Macromethod Wt. of Trypto.ample, phan,
hlicromethod Kt. of Tryptosample, phan, 116. % 0.5 1.22 1.0 1.30 1.0 1.36 0.5 6.30
F r o t a n Samplrq JIg. "6 1.24a Casein Egg albumin lo 1.19= Ribonuclease b 15 lo 1.33 Lysozyme€ 4 6.30 Staphylococcus a u r m s (strain 4A) 40 0.63 4.0 0.62 Sullivan a n d Hess (8) reported 1.24% for casein a n d 1.20% tryptophan for egg elhumin. b T h e authors are indebted t o M , Kunitz Rockefeller Institute for Medical Research Princeton N. J for crysialline ribonuclease and t o H. D. Lightbody, 'Western RLaion&"Resrarch Laboratory, Albany, Calif., f o r lysozyme sample.
The determination of the effect of endogenous heat on the maximal color development showed that in the macromethod there is sufficient heat (45" C.) liberated by the neutralization of slkali with the acid. The heat thus generated is Sufficient for 64 1
642
ANALYTICAL CHEMISTRY
These data shovi that a minimal temperature of 45” C. is necessary for the development of maximum color. and that the analysis of pure tryptophan requires an amino acid environment such as casein hydrolyzate, to prevent its destruction during alkaline treatment. Making use of these precautions, the results obtained with various materials are given in Table 111. Results would indicate that the modification of the method of Sullivan and Hess for the determination of tryptophan is adaptable to use with microquantities of protein.
(2) Bates, R. W., Proc. Am. SOC.B i d . Chem., J . Bid. Chem., 119, vii (1937). (3) Block, R. J., and Bolling D.. “Amino Acid Composition of Proteins and Foods,” Springfield,Ill., Charles C Thomas, 1945. (4) Gunness, M., Dwyer, I. M., and Stokes, J. L., J . B i d . Chem., 163, 159 (1945). (5) Kunita, M.,J . Gen. Physiol., 24, 15 (1940). (6) Swag, M .G., and Steers, E., Federation Proc., 7, 310 (1948). (7) Straus, E., Dingle, J. H., and Finland, &I., J . ImmirnoZ., 42, 331 (1941). (8) Sullivan, M. X., and Hess, IT. C., J . B i d . Chem., 155, 444 (1944)
LITERATURE C l T E D
(1) Alderton, G., Ward, IT. H., and Fevold, H. L., J . B i d . Chem., 157, 43 (1944).
RECEIVED April 13, 1948. Supported by a research grant from the Dilision of Research Grants and Fellowships, Sational Institute of Health, U. S. Public Health Service.
Spectrophotometric Determination of Iron in Ores with Kojic Acid J. P. MEHLIG AND M. J. SHEPHERD,
JR.
Ordcon State College, Corivdlis, Ore.
has pointed out that, in view of the very general MELLOX belief ’ that(8)colorimetric determinations are limited to maximuni concentrations of a few parts per million of the desired constituent, there is need for further study regarding the upper limit for reliable work viith modern instruments. This point of view deserves more attention than it usually receives. Some spectrophotometric work of this nature has been reported by the senior author and his co-workers (5-7). The purpose of the work described in this paper was to apply kojic acid, 5-hydroxy-2-(hydroxymethyl)-l,4-pyrone, to the spectrophotometric determination of iron in ores in an effort to furnish further proof that such determinations are not limited to a micro scale, but are practical for macroquantities as well. The reaction between kojic acid and ferric iron to produce a yellom-ish-orange complex found its first analytical application in the colorimetric determination of the acid with ferric chloride (1, IO). Moss and Mellon (9) reversed this process, using kojic acid as the reagent for the colorimetric determination of iron, and made a critical spectrophotometric study of the effect of diverse ions and other factors upon the color system. Kojic acid is the first pyrone derivative t o be used as a reagent for iron. APPARATUS AND SOLUTIONS
A11 spectrophotometric measurements were made with a CencoSheard spectrophotelometer. All p H measurements were made with a Beckman p H meter. An aqueous solution containing 1 grain of kojic acid per 1000 ml., a commercial 3y0 solution of hydrogen peroxide, and an aqueous solution containing 250 grams of ammonium acetate per 1000 ml. were used. Standard iron solutions, containing 1, 2, 3, 4, and 5 mg. of iron per 1000 ml., were prepared by dissolving the calculated weights of 99.95% ferrous ammonium su1fat)ehexahydrate in water, and adding 25 ml. of 12 M hydrochloric acid and enough hydrogen peroxide to ensure complete oxidation of the iron to the ferric condition. The excess of peroxide was decomposed by boiling and each solution after cooling LYas accurately diluted to 1000 ml. REFEREYCE CURVE
T o produce the color system 1 mI. of the appropriate standard solution of iron was carefully measured with a microburet into a 100ml. volumetric flask and 10 ml. of kojic acid solution were added, followed by 10 ml. of ammonium acetate solution to provide a pH value of 6.3 to 6.5. The reagents must be added in the order given; if the ammonium acetate solution is added before the kojic acid the reddish-brown color of ferric acetate interferes. The volume was then made up to 100 ml. .4 yellowish-orange color developed immediately. The transmittancy a t 440 mp (9)
was determined for each solution in a 1-em. cell. The entrance slit was set at a width of 2.5 mm. and the exit slit at 20 mp. T h e transmittancy was calculated by dividing the transmittancy of the standard solution by the transmittancy of the blank solvent. A reference curve was plotted correlating the logarithm of the extinction with the concentration of iron in milligrams per liter. No portion of this curve was a straight line. The deviation from a straight line was undoubtedly caused by the limitations of t h e instrument, as Moss and Mellon (9) have shown by using a General Electric spectrophotometer that the system does obey Beer’s. law for concentrations up to at least 20 mg. of iron per liter.
Table I. Sample To. 1 2 3 4
5 B 7 8 9 10 11
12 13
Results Obtained w - i t h Kqjic Acid Iron by Dichromate Xethod
Iron by I
