Manganaro of the Applied Research Laboratory, and C. S. Sheppard of the United States Steel Corp. Mellon Institute Project 237, for their advice and assistance during this work. LITERATURE CITED
(1) Ardashev, B. I., Trudy Ural. Ind.
Inst. im. 8. hf. Kirova 1938, s o . 6,
70-9. (2) Arreguine, Victor, Rev. univ. nacl. Cdjdoba ( A r g . )31, 1706 (1944). (3) CermBk, Miroslav, Chem. listy 45, 35-6 (1951). (4) Fleig, C., Compt. rend. SOC.biol. 6 5 , 283 (1908). (5) Fosse, R., Ann. chim. 6 , 13 (1916). (6) Gomberg, M., West, C. J., J . Am. Chem. SOC.34, 1529 (1912).
( 7 ) Haussler, E. P., Nitt. Gebiete Lebensm. Hyg. 38, 1 (1947). (8) Hayashi, Kaneo, J . SOC.7'rop. A g r . , Taihoku Imp. linio. 15, 20 (1943). (9) Sommer. L.. Chem. listu 47. 1415
(10) Wawzonek, S., in "Heterocyclic Compounds," R. C. Elderfield, Vol. 2, pp. 463-4, Wiley, New York, 1951. RECEIVED for review February 6, 1959. Accepted August 3, 1059.
Rapid and Specific Determination of Threonine MARTIN FLAVIN and CLARENCE SLAUGHTER Enzyme Section, National Heart Institute, Department of Health, Education, and Welfare, Bethesda,
b The acetaldehyde liberated by periodate oxidation of threonine may b e directly measured, after reduction of excess periodate with a mercaptan, by the amount of dihydrodiphosphopyridine nucleotide (DPNH) oxidized by alcohol dehydrogenase. The procedure requires 5 minutes, as compared with 2 to 5 hours for previous methods. All of the required reagents are available commercially.
P
specific methods for the determination of threonine, particularly in mixtures with other amino acids, have involved the diffusion, or forced aeration, of the acetaldehyde formed by periodate oxidation into trspping reagents such as bisulfite ( 8 , 9 ) , p-hydroxybiphenyl (9), or dimedon ( 5 , s - dimethyl - 1,3 - cyclohexanedione) ( 5 ) . The acetaldehyde is then usually measured by color formation with phydroxybiphenyl (or gravimetrically, in the case of the dimedon derivative).. These methods require 2 to 5 hours, and in the bitter two cases have been found unreproducible. h'either has it been possible to repeat a procedure for the direct measurement of the acetaldehyde with dinitrophenylhydrazine ( 7 ) . Now, however, the acetaldehyde can be measured in situ with D P K H and alcohol dehydrogenase, provided excess periodate is first reduced with a mercaptan. The method has been reproducible during constant use throughout the past y f a r in connection with studies of the mechanism of the enzymatic formation of threonine ( 4 ) . REVIOUS
REAGENTS
Molar potassium phosphate, p H 7.5, and 40/,sodium metaperiodate (Mallinckrodt analytical reagent grade) are prepared in distilled water, as are all other reagents. A 10% aqueous solu-
tion, by volume, of 3-mercaptopropionic acid (Eastman h'o. 6270) adjusted to p H 6 with potassium hydroxide is made up weekly, as longer storage leads to a decline in sulfhydryl titer ( 3 ) . This and the following reagents are kept at 0" C. while in use, and stored frozen. D P X H (Sigma Chemical Co., disodium) is oreoared as a 0.0025M solution. o H adjusied to 7 . 5 . Twice-crystallized yeast alcohol dehvdroeenase was obtained from the NutriGonal Biochemical Co. as a suapension in ammonium sulfate of 60 mg. of protein per ml., and was stored at -20" C. Though i t assayed a t only 40,000 units per mg. (I), this preparation has been stable and entirely satisfactory for a year. For use, several milliliters of a 100-to-1 dilution are prepared in the following diluent: 0.1% bovine serum albumin, 0.01M reduced neutral glutathione, and 0.02M potassium pyrophosphate, p H 7.5. PROCEDURE
The sample, containing 0.02 to 0.1 pmole of threonine, is pipetted into a 1-ml. volume, 1-cm. light path silica cuvette, followed b y 0.1 ml. of phosphate buffer and enough distilled water for a final volume of 1.0 ml. Three cuvettes are run at one time, against a water blank. After adding 0.02 ml. of periodate to the reaction cuvettes, the solutions are mixed well and allowed to react for 30 seconds. (The oxidation of threonine is incomplete in 15 seconds.) Then 0.03 ml. of mercaptopropionate is added, and the mixture again stirred for 30 seconds. After adding 0.04 or 0.05 ml. of D P N H , the solutions are again stirred, and two successive absorbance readings are made a t 340 mp, in the Beckman D U spectrophotometer, at room temperature (25" C.). These will show no decline in absorbance if reduction of periodate t o iodide b y the mercaptan has been complete. Finally, 0.02 or 0.03 ml. of alcohol dehydrogenase is added, and the oxidation of D P N H by acetaldehyde is fol-
Table I.
Md.
Specificity of Assay for Threonine
Ile!rease in Absorb-
Compounds Added uL-Threonine ~~-Allothreonine 2,3-Butaiiediol DL-Serine DLThreonine DL-serine Glucose Gl!m?rol DL-Homoserine 0-Phosphohomoserine DL-Aspartate DL-Methionine Tartrate DIrThreonine tartrate
+
+
Amount, ance, pmole 340 M p 0 1 0.40 0 1 0 41 0 05 0 35 0 1 0 1 0 0 0 1
1 0 0 1
0 40 0
0 1
0
0 1
0 0 0
0 1 0 1 0 1
0 5 0 1 0 5
0 0 0.34
lowed a t 340 mp until two successive readings a t half-minute intervals show no further decline in absorbance. The total decrease in absorbance, corrected for dilution by the enzyme, is the measure of the amount of threonine present. The amount of enzyme is sufficient to bring the rraction to completion in 1 to 3 minutes. The use of much larger amounts xould reduce the specificity of the assay ( I ) . RESULTS AND DISCUSSION
The change in absorbance at 340 mp is strictly proportional to the amount of threonine added, from 0.01 to 0.1 pmole. An occasional reference cuvette should be run with a known amount of threonine for, although the absorbance change per 0.1 pmole of threonine rc,mains constant with any one batch of reagents, it has varied, with different batches, between limits of 0.40 and 0.47. These values are about one third less than would be predicted from the absorptivity, 6300, of D P N H a t 340 mp. The explanation VOL. 31, NO. 12, DECEMBER I959
1983
for this discrepancy, which does not interfere with the assay, has not been ascertained. The other product of threonine oxidation, glyoxylate, is partially but not completely decomposed by neutral periodate in 30 seconds, and might inhibit alcohol dehydrogenase. As shown in Table I, some inhibition is observed if tartrate is added to the threonine sample. 2,3-ButanediolI which yields 2 moles of acetaldehyde, also gave less than theoretical change in absorbance, but the sample assayed was of unpurified technical grade. The commercial threonine samples used as standards gave approximately correct leucineequivalent values in quantitative ninhydrin tests (2), and paper chromatography has shown no ninhydrin-reactive components other than threonine. The p H chosen for the reaction is a compromise between those optimal for the periodate oxidation and the alcohol dehydrogenase reactions. The choice of 3-mercaptopropionate for periodate reduction was arbitrary; it was the first of the reducing agents tested which did not interfere with the subsequent oxidation of D P N H by alcohol dehydrogenase. Oxidation products of the mercaptan do retard the rate of the enzymatic reaction slightly, but this
Table II. Assay of 0.1 pmole of Threonine with Varying Amounts of Periodate and Mercaptan
Periodate, bll.
0.02 0.04
0.02 0.04
Decrease in Mercaptan, Absorbance, hll. 340 hfp 0.03 0.06 0.06 0.03
0.40
0.37 0.39 0.14
does not affect the usefulness of the assay. As shown in Table 11, the amounts of periodate and mercaptan added in the assay of 0.1 pmole of threonine are not highly critical. Still larger excesses of periodate will result in a n oxidation of D P N H before the alcohol dehydrogenase is added. Besides being rapid, sensitive, and reproducible, the assay has been quite specific for threonine. I n particular, Table I shows that compounds which yield formaldehyde with periodate do not interfere. Very few substances which are found naturally in appreciable amounts yield acetaldehyde on brief exposure to periodate, and rhamnose, the more widely occuring’ of these, besides threonine, is also usually present in high molecular weight polymers (6). The results of Table I11 suggest t h a t the method will also be applicable to the rapid estimation of threonine in hydrolyzates of 0.1-mg. amounts of proteins. The simulated hydrolyzate (Table 111) was a synthetic mixture obtained from the Beckman Instrument Co., diluted to contain 0.25 pmole per ml. of each of the following amino acids: lysine, histidine, arginine, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, 0.5 cystine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine. The acid-hydrolyzed casein was 2 commercial preparation. The crystalline pancreatic ribonuclease has been oxidized with performic acid, then hydrolyzed for 42 hours in 6N hydrochloric acid a t 110’ C. The hydrolyzstes were neutralized before asssy. The recoveries of known amounts of threonine added to aliquots of hydrolyzates are quantitative for each of the three preparations (Table 111). The recovery of hydrolyzate threonine was also proportional t o the size of the aliquot taken iE each case, although with the
casein i t was necessary to use twofold amounts of periodate and mercaptan to maintain proportionality with the largest aliquot. The values found for threonine correspond to theoretical for the simulated hydrolyzate, but, as would be expected from the known partial destruction of threonine during acid hydrolysis, are less than theory for casein [4.9% threonine (IO)]and ribonuclease (10 pmoles per pmole protein = 1.19 mg. per 14.58 mg. = 8.2%). Unfortunately, satisfactory threonine analyses by independent methods are not available for these hydrolyzates. The amounts of all the amino acids in the ribonuclease hydrolyzate were determined by the ninhydrin method ( d ) , after they had been separated from each other by ion exchange chromatography. By this method, recovery of threonine was 72.6y0of theory, and recoveries of the other amino acids were about 85%. If the low recoveries of acid-stable amino acids were due to errors in analysis rather than in preparation of the protein sample, the corrected threonine recovery would be 85%, as compared with 84y0 obtained with the present method (Table 111). Though not novel in principle, the speed of the assay has made it indispensable for the authors’ purposes. Its application under other conditions will be subject to the limitation of possible inhibition of the enzymatic reaction by some chemical contaminants. ACKNOWLEDGMENT
The authors are indebted to Juanita Cooke of the Laboratory of Cell Physiology, National Heart Institute, for providing the ribonuclease hydrolyzate and the results of the ninhydrin analyses thereof. LITERATURE CITED
Table 111.
(1) Colowick, S. P., Kaplan, S. O.,
Threonine Assay in Protein Hydrolyzates
De-
crease in
Ab-
Hydrolyzate
“Methods in Enzymology,” Vol. I, p. 500, Academic Press, New York,
Recovery
19.5.5. -I_-.
of
Threo- sorb- Added Hydrolyzate Threonine, nine ance, Threopmole Added, 340 nine, Amount pmole Mp 70 \i Found Calcd. 0- ..4- 5_
Simulated hydrolyzate, ml.
0.1 0.2 0.4
Casein, acid hydrolyzate, mg.
0.1 0.13 0.25 0.50 0.50“
Ribonuclease, acid hydrolyzate, mp.
0.13 0 074
0 11 0 15 0 074
Assay with twofold amounts of periodate
1984
ANALYTICAL CHEMISTRY
0.1
0.05
0.05
0.11
0 22
0.42 0.33 0.12 0.24 0.36 0.49 0.36
19 29 39 0 05 41 and mercaptan. 0 0 0 0
98
107 98
0.024 0.048 0.093
0.025 0.050 0.10
0.027 0.053 0.080 0.109
0.053 0.103 0.205 0.205
0 042 0 064 0 087
0 051 0 075 0 103
(2) Ibid., Vol. 111, p. 503, 1957. (3) Ellman, G. L., Arch. Biochem. Biophys. 74, 443 (1958). (4) . , Flavin. M.. Slaughter. C.. Federation Proc. 18: 226 (195g). (5) Gaffnei, G.‘ W.,’ W‘illiams, W. A., McKennis, H., Jr., ANAL. CHEX 26,
___
588 f,----,. 19.54)
(6) Huggins, C. G., Miller, 0. Y., J . Biol. Chem. 221, 377 (1956). (7) Karasi Ibid., 227, 1
. &iochem.’J. 35 , 294 (194i).’ (9) Neidig, B. A., Hess, W. c . , ANAL. CHEM.24, 1627 (1952). (10). N y r a t h , H., Bailey, K., “The Proteins, Vol. I, Chap. 3, Academic Press, New York, 1953. RECEIVEDfor review June 11, 1959. Accepted September 15, 1959.
