Simultaneous Estimation of Threonine and Serine B. A. NEIDIG A h D W. C. HESS Department of Biological Chemistry, Georgetown CTnit.ersitySchool of Medicine and Dentistry, Washington 7 , D. C . LOCK and Bolling ( 3 ) were the first to determine threonine by oxidation to acetaldehyde, employing lead tetraacetate as the oxidant. I n 1941, Shinn and Kicolet determined threonine (10) and serine (8) by oxidation with periodic acid. The acetaldehyde yielded by threonine was aerated into a sodium bisulfite solution, and mas estimated by titration of the bisulfite complex with iodine after the oxidation of the excess bisulfite. The formaldehyde formed from the serine remained within the oxidizing mixture, and was quantitatively precipitated with dimedon. Eegriwe (5) found that acetaldehyde reacted with p-hydroxybiphenyl to produce a red- violet^ colored compound, and later (6) that formaldehyde reacted with 1,8-dihydroxynaphthalene-3,6disulfonic acid, chromotropic avid, to produce a rose-colored compound. The former colorimetric method had been used by Block and Boiling (9) for the estimation of the acetaldehyde formed from threonine. Chromotropic acid was used by Boyd and Logan (4) in the determination of the formaldehyde produced by the periodic acid oxidation of serine. Rees (9) proposed the simultaneous determination of threonine and serine by oxidation with periodic acid and aeration of the acetaldehyde followed by the distillation of the formaldehyde into sodium bisulfite solutions. Both aldehyde bisulfite complexes were finally estimated by iodoinetric titration. In the following experiments simultaneous iiiicrodeterniinations of threonine and serine are effected using periodic acid for oxidation, but substituting the more sensitive colorimetric methods of Eegriwe (5, 6) for the determinations of the aldehydes. Several proteins were analyzed by the procedure developed, and the values obtained are given. RE4GEiYTS
Borate buffer, pH 8. T o 50 ml. of 0.2 Jf potassium chloride are added 3.97 ml. of 0.2 S sodium hydroxide and the solution is diluted to 200 ml. Aqueous periodic acid, 59;. Sodium hydroxide, 0.2 S . Sodium bisulfite, 2% Copper sulfate, 4%. p-Hydroxybiphenyl, l.SCc, dissolved in 0.5% sodium hydroxide. Sulfuric acid. 1 to 3. Sodium arsenite, 0.1 S,dissolved in 100 ml. of distilled water containing 2 grams of sodium bicarbonate. Aqueous chromotropic acid, 5%. It was found that this reagent decomposed rapidly on standing; however, if l gram of norite was added to 25 ml. of thr aqueous solution and then filtered, the filtrate was stahlr foi a t least a weeh if kept in a refrigerator. EXPERIMEhTAL PROCEDURE
Oxidation of Serine and Threonine. An oxidation and aeration train is prepared by connecting in series a sulfuric acid drying flask, three large 20 X 2.5 em. test tubes, and finally a suction trap leading to a filter pump. In the first test tube, adjacent to the drying flask, is placed 5 ml. of a solution containing from 20 to 100 micrograms each of serine and threonine. Tubes 2 and 3 each contain 5 ml. of 2T0 sodium bisulfite, and are cooled in an ice bath. To tube 1 are added 15 ml. of borate buffer, 1 ml. of 5 r o periodic acid, and 2 ml. of 0.2 S sodium hydroxide. A fairly rapid flow of air through the system is maintained for 1 hour. Tubes 2 and 3 are removed, their contents combined and allowed to stand a t room temperature for a t least 1 hour prior to acetaldehyde estimation. The contents of tube 1 are transferred to a 250-ml. Claissen flask and reseived for formaldehyde estimation. Determination of Acetaldehyde from Threonine. -1 1-nil. aliquot from the combined contents of tubes 2 and 3 is placed in a large test tube, 1 drop of 4% copper sulfate added n i t h shaking, followed by 6 ml. of concentrated sulfuric acid. The tube must be shaken continuously during this addition. After cooling in an ice bath, 0.2 nil. of the p-hydroxybiphenyl solution is added, and the tube is kept a t 37" for a hdf hour and then placed in a boiling
water bath for 1.5 minutes to remove any excess solid p-hydroxybiphenyl. The tube is then allowed to cool to room temperature and the violet color read in a photoelectric colorimeter using a 540 filter. A distilled water blank is prepared similarly. An alternative method for the estimation of the acetaldehyde may be employed in which tube 3 of the oxidation train is omitted and in tube 2 are introduced 1 ml. of distilled water, 1 drop of 4% copper sulfate, and 6 ml. of concentrated sulfuric acid. The tube is cooled and 0.2 ml. of p-hydroxybiphenyl is added and placed in the train. Oxidation and aeration are conducted as previously described, and, a t the end of the aeration, test tube 2 is placed in a boiling water bath for 1.5 minutes, cooled, and read as before. This method permits the use of 2 to 10 micrograms of threonine in the sample tube instead of the 20 to 100 micrograms for the above described procedure.
Table I.
Experimental Values for Threonine and Serine
(Percentages, corrected to 16% nitrogen, are conipared with values obtained from Block and Bolling) Threonine Sprine ____ Protein %N Expt. Lit. Expt. Lit. Egg albumin 12.11 3.7 3 1-4 3 8.5 7.6-8.3 Casein 15.37 4.6 3.6G4.8 5.4 5.7-7.5 Dentine 16.15 3.1 ., . . , .. 6.7 .. , . . .. Edestin 18.40 3.0 3.3 3.1 5,4 Gelatin 17.60 2.2 1.4-2.0 4.2 3.3 Human globin 14.51 5.7 6 8 6.4 ... Squash seed 16.34 3.0 5.6 Zein 15.70 3.0 2.iI3 4 6 2 7.0l7.8
Estimation of Formaldehyde. T o the contents of the Claissen flask are added 5 ml. of water, 10 ml. of the arsenite solution to reduce the unreacted periodic acid, and several drops of bromocresol green. The p H is carefully adjusted by the dropwise addition of 1 to 3 sulfuric acid until the solution just turns yellow, about 6 drops being required; then arsenite solution is added, dropwise, until the solution becomes green, pH 4.8; and finally 1 ml. of excess arsenite is added. The acidity must be carefully adjusted, for if the solution is too alkaline the formaldehyde will not distill, and if too acid free iodine is liberated, which passes into the distillate and interferes with the colorimetric determination. The contents of the Claissen flask are then distilled directly into 10 ml. of O.27@sodium bisulfite in a 50-ml. graduated cylinder. After 40 ml. have been rollected, the distillation is stopped, the volume is adjusted t o 50 ml., and the contents are well mixed. From this distillate, a 5-ml. aliquot, containing formaldehyde equivalent to 2 to 10 micrograms of serine, is placed in a large test tube and 4 ml. of concentrated sulfuric acid are added with continuous shaking, and the mixture is cooled in an ice bath. To the solution is then added 0.1 ml. of 5 7 , chromotropic acid, and it is placed in a boiling nater bath for 30 minutes. After cooling to room temperature it is read in a photoelectric colorimeter using a 540 filter. A distilled water blank is prepared similarly. Preparation of the Protein Hydrolyzate. The hydrolyzate was prepared by heating 100 mg. of the protein with 5 ml. of 2 0 7 hydrochloric acid in a wax bath a t 120" to 125' C. for 6 hours. The hydrolyzate was neutralized with 5 'V sodium hydroxide, dropwise with stirring, to about pH 3.5 and diluted with distilled water to 50 ml. One milliliter of the solution contained 2 mg. of protein. Accuracy of the Method. Aqueous solutions of DL-threonine and DL-serine, containing 20 mg. of each amino acid, were prepared. A 10-ml. aliquot was diluted to 100 nil. and 5-ml. aliquots of the final solution, containing 100 micrograms of threonine and serine, were used for the determinations. Four separate samples were taken and five different determinations were made on each. The average colorimeter readings, corrected for blanks, were calculated for both threonine and serine and the values obtained plotted. The individual values obtained were also included to show the variation from the average values for the several concentrations employed. The coefficients of variability, based upon the standard deviation for each amino acid, were determined to be 5.9% for threonine and 7 . 7 7 , for serine. These values give the maximum variations, whereas the means fall upon a straight line. Application to Proteins. The following proteins were analyzed
1627
1628
A N A L Y T I CA L C H E M I S T R Y
for their threonine and serine content: casein, squash seed globulin, dentinal protein, and edestin, which were prepared in this laboratory; egg albumin, obtained from J. B. Allison of Rutgers University; granular gelatin, U.S.P.; human globin hydrochloride from Sharp & Dohme; and alkali-precipitated zein from the Corn Products Refining Co. The average of the results, expressed as per cent, and based on 1670 nitrogen appear in Table I. The values obtained are compared with the ranges of values for similar proteins as given by Block and Bolling (1, 2 ) . DISCUSSION
The use of colorimetric methods for the estimation of the acetaldehyde and formaldehyde produced by periodic acid oxidation of threonine and serine permits the determination to be made upon smaller samples than those originally employed by Shinn and Nicolet (8, 10) and in a shorter period of time. By the colorimetric methods only a single oxidation is necessary. The results obtained upon samples of known concentrations of both amino acids have been graphed, and show that an average of four values falls upon a straight line, although there is a deviation of from 6 to 9% for single determinations a t each concentration. The values given for a number of proteins are in agreement with the literature values for the same proteins using the original methods of Shinn and Kicolet. The same criticism of the serine method originally noted by Nicolet and Shinn ( 8 ) )that hydroxylysine will interfere, still holds with the colorimetric method. Among the proteins analyzed by the authors, as far as present knowledge goes, only gelatin contains hydroxylysine. Inskip ( 7 ) has not found any of the
other proteins to contain this amino acid. I n determining the amount of serine present in a gelatin hydrolyzate a correction hae been made for the hydroxylysine present. The alternate method for the determination of threonine, in which the acetaldehyde is aerated directly into the color reagent, p-hydroxybiphenyl, a8 suggested by Block and Bolling (Z), requires less time and there is somewhat less variation in the colorimetric readings a t each concentration than in the procedure first described. LITERATURE CITED
(1) Block, R. J., a n d Bolling, D i a n a , "Amino Acid Composition of Proteins a n d Foods," 2nd ed., Springfield, Ill., C. C T h o m a s Publishers, 1951. (2) Block, R. J., a n d Bolling, D i a n a , "Determination of t h e Amino Acids," Revised e d . , Minneapolis, M i n n , , Burgess Publishing Co., 1940. (3) Block, R. J., a n d Bolling. D i a n a , J . Biol. Chem., 130, 365 (1939). (4) Boyd, H. J., a n d Logan, AI. A., Ihid.,146,279 (1942). (5) Eegriwe, Edn-in, 2. anal. Chem., 95, 323 (1933). (6) Eegriwe, Edwin, Ibid., 110, 22 (1937). (7) Inskip, L. W., J . Am. Chem. Soc., 73,5463 (1951). (8) Kicolet, B. E . , a n d Shinn, L. 9..J . Bid. Chem., 139, 687 (1941). (9) Rees, hI. IT., Biochem. J.,40, 632 (1946). (10) Shinn, L. A, a n d Kicolet, B. E., J . Riol. Chem., 139, 687 (1941).
HECEIVED for review M a y 9, 1952. Accepted June 14, 1952. These studies were substantially aided by Contract K R 181-817 between the Office of Naval Research, Department of the S a w , and Georgetown University, Material taken, in part, from a thesis submitted by the senior author t o the Graduate School of Georgetown University in partial fulfillment of the requirements for the degree of master oi science.
Rapid Method for Paper Chromatography of Organic Acids F. W. DENISON,
JR., AND
E. F. PHARES
Biology Disision, Oak Ridge National Laboratory, Oak Ridge, Tenn.
OST methods for the paper chromatography of organic acids employ slom-traveling solvents and long-drying procedures (1.3,6)requiring many hours for completion of the chromatogram, The method described in this paper using ether, acetic or formic acid, and water, requires less than 2 hours for satisfactory development of the chromatogram. The speed of development is slightly faster than the chloroform and iso-octane solvents described by Stark, Goodban, and Owens ( 3 ) . Only 10 to 15 minutes are required to free the paper of siiamping acid. The acids may bP applied as the free acids in ivater or organic solvents, or as a solution of the sodium salts, acidified with sulfuric acid either before or after application to the paper. The general technique is similar to that of Lugg and Overell ( 1 ) but offers advantages in simplicity and rapidity. The method should be of value for a rapid survey of unknown acids and especially for more positive identification when used in conjunction with other solvent systems (1, S), and mith partition columns, on uhich the relative positions of the acids are changed considerably ( 2 ) . METHOD
Approximately 0.0002 meq. of the organic acids in nater or organic solvent x a s applied to 40 X 57 em. sheets of Whatman No. 1 paper in spots 4 em. apart and 3 em. from the bottom of the paper. The spots were kept to a diameter of less than 1 em. by the repeated application of less than 5-microliter amounts, the drying process being aided by a stream of warm air from a small hair dryer. The paper was stapled into the form of a cylinder, leaving a small space between the edges of the paper, and placed in a glass jar, the bottom of which was covered to a depth of about 1 cm. with the solvent solution. The top of the jar was covered with a weighted glass plate, using a neoprene gasket to effect an air-tight seal. FVith a temperature of 22" C. the solvent front traveled about 15 cm. above the initial spots
in 1.5 hours. At the end of the development period the paper was suspended in a hood. -2 steam manifold, 60 em. in length with holes about 1 cm. in diameter and spaced about 10 em. apart, was placed about 25 em. in front of the paper and a strong flow of steam tc as maintained over the entire surface of the paper. An infrared lamp was focused on each half of the paper from a distance of about 40 em. K i t h this treatment the acetic acid was removed in 5 to 10 minutes and formic acid in 15 minutes. The chromatogram was then sprayed M ith neutral or slightly alkaline bromocresol green, 400 mg. per liter of 95% ethyl alcohol. R, values were measured fiom the center of the acid spots. The Rj values obtained by applying solutions of sodium salts (Table I, column E ) were lower than those from the free acids. This \\as overcome by acidification of the solution with sulfuric acid to a pH of about 1. Considerably higher concentration of acid did not appear harmful. Spots of sodium salts were also acidified on the paper by addition of small amounts of dilute
Table I. HI X 100 for Organic Acids Acid A B C D E .. 9 .. Tartaric .. 12 11 19 43 0 Oxalic 71 08 Citric 16 08 06 24 78 Malic 13 20 25 16 71 51 36 a-Ke toglutaric 14 82 43 45 49 28 Glycolic 82 32 40 73 52 Malonic 78 45 91 21 and 56 45 Pyruvic 70 87 87 72 58 Lactic 93 75 8.5 62 Succinic 76 90 94 98 84 90 Fumaric Oxalacetic 46 A = 13 parts ether, 3 parts glacial acetic, 1 part water. B = 14 parts ether 1 part glacial acetic, 1 part water. C = 1 4 partq ether' 0.2 part formic 1.8 parts water. D = 6 partsacetone 6 parts ether 3 parts glacial acetic, 1 part water. E = Same as A , but'sodium salts k e d for spotting with no sulfuric acid.
