V O L U M E 28, NO. 2, F E B R U A R Y 1 9 5 6 tion of an unknown mixture, it is advisable to separate the 2-cyanopyridine fraction by distillation as described elsewhere
(4). ACKNOWLEDG\IENT
Support of this work by a grant-in-aid from the Xational Science Foundation is gratefully acknowledged. The purchase of the Sargent pen recording polarograph was made possible through nn apparatus grant to one of the authors (R.-i.O.) by Trustees Research Committee, Rensselaer Polytechnic Institute The authors wish to thank S. C. Waite. J r . , for assistance in sonip of the measurements.
265 LITERATURE CITED
J.,and Janz. G. J.,J . Am. Cheiii. Soc. 7 4 , 1790 (1962). (2) Janz, G. J., and Hawkins, P. J.,.Vatwe 162, 28 (1948). (3) Jans, G. J.,Alscah,K. G., and Keenan, A. G.. Can. J . Research B25,272 (1947). (4) Jarvie, J. A I . S., Fitsgerald. W. E , and Janz, G . J..,J, d r r i . C ' h e r r ~ . Soc., in press. (5) Knobloch, E., Collection Czechosloc. C h e m . Corrirrutris. 12, 407 (1947). (e) Tompkins, C., and Schmidt, C. L. A , . r n i o . Calif. ( B e r k e l e y ) Publs. Phusiol. 8, 229 (1944). (1) Hawkins, P.
R E C E I V E Dfor r e v i m J u n e 1, 1925.
Accepted Soveinher 10. 19%
Colorimetric Ninhydrin Reaction for Measlrrement of Alpha-Amino Nitrogen HAROLD KALANT Department o f Pathological Chemistry, University o f Toronto, Toronto, Canada
The photometric ninhydrin reaction of Cocking and Yemm can be applied safely to the measurement of free amino nitrogen in tungstic acid filtrates of plasma. Excess of ninhydrin or of cyanide reagent exerts a deleterious effect on color development, as does the presence of peroxides. hletal salts were not found to interfere when a citrate buffer was employed. Other protein piecipitants tested were not found satisfactory. A r k rial plasma from normal fasted dogs gave an average valiie of 4.91 mg. per 100 ml.
C
OLORIMETRIC measurement of a-amino nitrogen by means of the purple color formed on reaction with ninhydrin was originally proposed by Harding and 3lacLean ( 2 ) , arid first placed on a sound quantitative basis by Moore and Stein ( 5 ) . This procedure has undergone n ~ ~ m e r o uimprovements s in recent years. T h e use of reducing agents such as stannous chloride ( 5 ) , ascorbic acid ( I O ) , potassium cyanide (1, 9, If), or reduced ninhydrin ( 6 ) , and addition of various organic solvents such as pyridine (9, 10)or methyl Cellosolve ( I , 5 ) t o the reaction mixtiire result in uniform xield of the same colored product for equiniolecular amounts of almost all of the a-amino acids. An adequate buffer, generally a citrate buffer a t pH 5 (1, 4,5 ) , permits i!se of the method with biological fluids or protein hydrolyrates. The method of Cocking and Teinm ( 1 , 11), which incorporates all these features, has been employed in this laboratory t)ecnuse of its simplicity and accuracy. INFLUENCE O F PEROXIDES
Temm and Cocking ( 1 1 ) specify that the methyl Cellosolve employed should give a faint or negative peroxide test with 10% potassium iodide. The importance of this warning is shown in the following observations. After a long period of successful use of the method as described by the authors, it was found that the reference standard solutions of pure amino acids had begun t o j-ield progressively less color. This decrease became increasingly rapid, until after a month the absorbance was only 15% of its usual value. At first, increasing the amount of ninhydrin reagent above the recommended quantity of 0.2 ml. restored the full color yield, but later, amounts of 1.5 or even 2.0 nil. failed to do PO.
The meth)-l C~elloeolveused in preparation of the ninhydrin :1nd cyanide reagent E had been originally redistilled and stored in a dark bottle. It was n o x found, however, t o have developed a high content of peroxide as indicated by liberation of free iodine from acidified potassiiirn iodide solution, decolorization of I h r k oside of nickel, Prussian blue test, and blackening of inetnllic~ mercury. Tests for reactive carbonyl groups Tere ncgntive. After distillation from a zinc-copper couple the Cellosolve was fi,ee of peroxide, and reagent solutions made with it p1r.e the. iisual fiill color yield in the ninhydrin reaction. Hydroquinone added to the stock reagents in concentrations of 0.001 t o 0.01% does not impair the color reaction, and may serve to prevent peroxide formation. Storage of the Cellosolve under nitrogen, or in small filled dark bottles. is proh:tbl>. also advisable. INFLUENCE OF CY 4YIDE CONCENTR 4TIO\
In view of the harmful effect of peroxides, the influence of variation in the amount of cyanide reagent was determined t - q ing replicate samples of a standard amino acid solution, the reaction was carried out Kith 0.5, 1.0, 1.5, and 2.0 ml. of 2 x 10-4.1P cyanide-Cellosolve reagent. All reaction nii.ctiirrq were
Table I.
Effect of Added Substances on Colorimetric Ninhydrin Reaction
Added hfaterial Zl-one Cobalt nitrate Copper sulfate Ferrous sulfate Versene (satd. rollition in 0 2.11 citrate buffer, p H 5 )
Absorbance 0 208 0 201 0 248 0 ?Oli 0 217
Each reaction mixture contained 5.4 y of a-amino nitrowen. .-\brorhance of each was determined by comparison with a reagent bTank containin. the same added material.
made t o the same volume with meth:.l Cellosolve before heating. \Then peroxide-free reagents were employed, mavimal color yield was obtained with all but the smallest amount of cyanide reagent. When the reagents were made with Cellosolve contaminated with peroxides, use of increasing amounts of cyanide
266
ANALYTICAL CHEMISTRY
reagent resulted in a progressive increase in color yield, approaching maximal values with 2.0 ml. of cyanide reagent. The use of 1.0 ml. of 2 X 10-3M cyanide reagent, however, resulted in a deep orange-brown color in the reagent blank, which grew steadily more intense over a 20-minute period following the heating. Amino acid reaction mixtures prepared with the 2 'X l O - 3 X cyanide reagent showed a color which appeared to be the resultant of this orange-brown color and Ruhemann's purple. When compared photometrically x i t h the blank, using the Evelyn 575 filter, the reaction mixtures showed a rapid progressive decrease in absorbance which was really due t o the increase in the blank reading. This suggests that the excess cyanide reacts with ninhydrin, producing the orange colored substance with measurable absorbance a t 575 mp. Since less ninhydrin
correlation between color yield and amount of ninhydrin. Amounts larger than the recommended 0.2 ml. of 5% solution were found to inhibit color development. Lea andRhodes (3)have called attention to the inhibitory effect of an increased proportion of Cellosolve in the reaction mixture. I n the present case, however, as the same water-Cellosolve ratio was present in all tubes, the inhibitory effects observed m-ith larger amounts of ninhydrin reagent may be attributed to the excess of ninhydrin itself. No ready explanation is a t hand for this fact, but the finding is in conformity with the suggestion of RIoubasher ( 7 ) that the amine must be present in excess of the reduction product of ninhydrin, for maximal color yield. The inhibitory effect of larger quantities was not observed with ninhydrin reagent contaminated with peroxides. The peroxides appear to have SO reduced the effective concentration of ninhydrin, that the effective quantities of reactant used were all on the ascending limb of the curve in Figure 1. APPLICATION TO DEPROTEINIZED PLASMA
0 0 5 0.1 0 2 0.5
1.0
ml. ninhydrin Figure 1. Effect of varying amounts of ninhydrin reagent on color development with 5 y of a-amino nitrogen
remains in the test mixtures than in the reagent blank, this superimposed color is less, the larger the amount of amino nitrogen in the sample. Therefore, proportional readings cannot be obtained in the presence of such an excess of cyanide. INFLUENCE O F METALLIC CONTAMINANTS
Meyer and Riklis ( 4 ) have suggested that various cations impair the ninhydrin reaction with amino acids. The influence of such contaminants was tested in the Cocking and Yemm method, Milligram quantities of several salts and of Versene [(ethylenedinitrilo) tetraacetic acid] were added to standard reaction mixtures and corresponding blanks, prior to heating. The results shown in Table I indicate that in the presence of citrate buffer the added salts were without inhibitory effect; if anything, copper sulfate appeared t o improve color development slightly. Versene was also without effect. lleyer and Riklis added the metallic contaminants to unbuffered reaction mixtures; the Versene which they used was dissolved in citrate buffer. It is possible, therefore, that the beneficial effect noted by them was due t o the citrate buffer rather than to the Versene. INFLUENCE O F NINHYDRIh- CONCENTRATION
Using reagents made tvith peroxide-free methyl Cellosolve, a series of equal samples of standard aspartic acid solution and corresponding blanks were subjected to reaction with various amounts of ninhydrin reagent. -411 were made t o the same volume with methyl Cellosolve before heating, so that the same water-Cellosolve ratio obtained in all tubes. Figure 1 shows the
The ninhydrin reaction of Cocking and Yemm was applied to samples of plasma which had been deproteinized by ultrafiltration under positive pressure through a cellulose membrane, and precipitation with several protein precipitants. Ultrafiltration was found to yield very variable results, due to small quantities of protein escaping through the filter. This is in agreement with the findings of Stein and hIoore (8). Ethyl alcohol, 1% tungstic acid, and 5% trichloroacetic acid, when added in 0.2-ml. quantities t o standard reaction mixtures and blanks, did not appear to affect color development; 5% sulfosalicylic acid in the same amount inhibited it completely. When the former three agents were used for deproteinizing plasma, however, only tungstic acid proved satisfactory. Ethyl alcohol did not give complete deproteinization a t concentrations low enough to permit most of the amino acids t o remain in solution. Lse of trichloroacetic acid resulted in the development of a sherry color, rather than the purple color normally given by amino acids. Recovery of added amino acids from plasma samples precipitated with nine volumes of 1% tungstic acid was 97 i 4% (extremes of range). Nine samples of arterial plasma from normal dogs in the postabsorptive state showed amino nitrogen concentrations ranging from 4.11 to 5.48 mg. per 100 ml., with a mean and standard deviation of 4.91 & 0.45 mg. per 100 ml. The corresponding venous levels ranged from 4.03 t o 5.49, with a mean and standard deviation of 4.72 0.46 mg. per 100 ml. ACKNOWLEDGMENT
This work was carried out during tenure of a Senior RIedical Research Fellowship of the National Research Council of Canada. LITERATURE CITED
Cocking, E. C., and Yemm, E. W., Biochenz. J . 58, xii (1954). Harding, V. J., and UacLean, K. AI., J . B i d . Chent. 20, 217 ( 19 15).
Lea, C. H., and Rhodes, D. h-., Bwchim. et Biophys. Acta 17,416 (1955).
AIeyer, H., and Riklis, E., Sattrre 172,543 (1953). Moore, S., and Stein, IT-. H.. J . B i d . Chem. 176,367 (1948). Zbid., 211, 907 (1954). Noubasher, R..Ibid., 175, 187 (1948). Stein, 1%'. H., and Moore, S.,Zbid., 211, 915 (1954). Troll, W., and Cannan, R. K., Ihid., 200, 803 (1953). Yamagishi, 1I.,and Yoshida, T., J . Pharm. SOC.Japan 73, 675 (1953). Yemm, E. W., and Cocking, E. C., Analyst 80,209 (1955).
R E C E I V Efor D review July 20, 1955,
Accepted October 26, 1955.
