An improved colorimetric determination of amino acids with the use of ninhydrin

ANALYTICAL

An

14, 71-77 (1966)

BIOCHEMISTRY

Improved

Calorimetric with the

YA PIN From

LEE

AND

Determination Use of Ninhydrin TUNEKAZU

August

Acids

TAKAHASHI

the Department of Biochemistry, Guy and Bertha Laboratory, University of North Dakota, School Grand Forks, North Dakota

Received

of Amino

Ireland Research of Medicine,

3, 1965

Attempts have been made to develop a simpler and more specific method for determining free a-amino acids in chemical and biological systems. The results obtained are presented in this paper. METHODS

AND

RESULTS

0.5 M citrate (Na+) buffer was prepared from analyticalgrade citric acid which was free of ammonia or amino acids. pH of the citrate buffer was adjusted to a desired value. A 1% ninhydrin solution was prepared by dissolving 1 gm of ninhydrin in 0.5M citrate buffer solution. Commercial ninhydrin preparations were found to be satisfactory without further purification. Some commercial glycerol preparations which were found to interfere with color development or to give a high blank reading were diluted with an equal amount of water to 50% (v/v) solution. The diluted solution was passed through a column of mixed resins of Amberlite ion exchanger to remove the contaminants. The effluent was concentrated to the anhydrous state (approximately 99.5%). Most of the amino acids, which were chromatographically pure, were obtained from Mann Research Laboratories, Inc., New York City. Amino acids, amines, or other compounds were dissolved in water, dilute NaOH, or dilute HCl solution. Procedure: 1.0 ml of a reaction mixture which consisted of citrate buffer, ninhydrin, and glycerol was pipetted into a test tube. Less than or 1 ml of sample and additional compounds to be tested, if there was any, were introduced into the reaction mixture. The total volume was made up to 2.0 ml with water. After shaking, the test tube was placed in a boiling water bath for a certain time as described in each experiment. After cooling in a tap-water bath at room temperature, a reading was taken at 570 rnp within an hour. The reagent blank and standard amino Reagents:

71

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acid solution assay.

AND

TAKAHASHI

were run at the same time to verify

and standardize

the

Organic solvents such as ethano1, dioxane, methyl Cellosolve, pyridine, and phenol were foimd to accelerate the color development to some extent (l-6). There are several unsatisfactory points in using these solvents because of volatility, unpleasant odor, or instability. It was therefore, desirable to use other substances to substitute for these solvents to improve the color development. Glycerol was found to be a better medium for the color development. The color reached its maximal intensity at 55% (v/v). The blank reading approximately doubled at 65% of glycerol and rose continuously (Fig. 1). Glycerol

Concentration

0

IO

and Color Development:

20 Glycerol

30 Concentration

40 (per

50 cent

60

70

v/v)

FIG. 1. Glycerol concentration and color development: (A) reagent blank; (0) sample-reagent blank. The reaction mixture (2 ml), which consists of 0.2 amole tryptophan, 0.35M citrate, 5 mg ninhydrin, and glycerol, was heated in a boiling water bath for 10 min. The fmal pH of the reaction mixture was 6.0.

Ninhydrin

Color development concentration up to 5 mg per blank reading was relatively of the reaction mixture under curve was obtained when the and pH value 5.5. reactions were conducted in

Concentration and Color Development:

was relatively proportional to the ninhydrin 2 ml of the reaction mixture (Fig. 2). The more sharply increased after 5 mg per 2 ml the experimental conditions. A quite similar final glycerol concentration was 25% (v/v) pH and Color Development: When the

AMINO

ACID

Ninhydrm

73

DETERMINATION

Concentration

(Mg 12ml)

2. Ninhydrin concentration and color development: (A) reagent blank; (0) sample-reagent blank. The reaction mixture (2 ml), which consists of 0.1 pmole L-tryptophan, 0.35 M citrate buffer, 60% (v/v) glycerol, and ninhydrin, was heated in a boiling water bath for 10 min. The final pH of the reaction mixtures was between 5.7 and 5.75. FIG.

h

I 6.0

6.5

7.0

I 7.5

PH

FIG. 3. Color development as a function of pH. The reaction mixture (2 ml), which consists of 0.1 &mole of compound, 5 mg ninhydrin, 0.35 M citrate, 60% (v/v) glycerol, and glycylglycine (0)) L-histidine (a), DL-valine (Cl), or epinephrine (A), was heated in a boiling water bath for 10 min.

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60% glycerol, the color development was at its maximum around pH 6.2 as shown in Fig. 3. It seems that the color yield as a function of pH is dependent upon the individual compounds. Heating Time and Color Development: Color development for most of the amino acids reached the maximal intensity in 15 min, thereafter the rate of color development was small under the experimental conditions (Fig. 4). Several amino acids were found to react at a relatively slower

0

I

2

3

4

5

6

7

6

9

IO

II

12

13

14

15

Minutes

4. Heating time and color development. The reaction mixture (2 ml), which consists of 0.35 M citrate, 5 mg ninhydrin, 60% (v/v) glycerol, and 0.1 /Imole amino acid except tryptophan (O), was heated in a boiling water bath for a certain time. Reagent blank, urea, or uric acid (A 1, nn-valine ( X ), L-histidine (A), phenylalanine or L-alanine (‘0)) n-aspartic acid ( W), glycine or tyrosine (a). FIQ.

rate with ninhydrin, and color intensity intensity in the presence of urea or uric reagent blank. Stability of Color Intensity: The color the reaction was conducted in higher About 1% of the color intensity faded 25% glycerol concentration. The blank should be, therefore, taken within 1 hr.

was slightly lower. The color acid was similar to that of the intensity was quite stable when glycerol concentration (60%). in 1 hr compared with 10% in color also decreased. Readings

Protein Precipitants and Color Development: Under our experimental

conditions perchloric

as shown in Table 1, metaphosphoric, trichloroacetic, or acids did not modify the color development, nor the intensity

AMINO

ACID

DETERMINATION

TABLE EFFECT

OF METAPHOSPHORIC, ON COLOR

75

1

TRICHLOROACETIC, DEVELOPMENT

OR PERCHLORIC

ACID

OD at 570 m&i Acid added

None Metaphosphoric Trichloroacetic Perchloric

Final

6.1 6.05 6.0 6.0

pH

Blsnk

Sample

Difference

0.12 0.12 0.125 0.121

1.044 1.04 1.05 1.06

0.924 0.92 0.925 0.939

The reaction mixture consists of 5 mg ninhydrin, tryptophan, glycerol (60y0’,), and the addition. The boiling water bath for 10 min.

0.35 M citrate reaction mixtures

buffer, 0.2 pmole were heated in a

of the reagent blank color when the pH value of the reaction mixture was kept constant. If the alkalinity or acidity of samples is too strong compared with the buffer capacity in the reaction mixture, the samples should be neutralized before adding to the reaction mixture. In order to avoid the shift of pH value, higher citrate concentration was used in routine assays. Amino Acid Concentration and Color Development: Color development was proportional to the concentration of amino acid present in the reaction mixture (Fig. 5). The color yield by glycylglycine or ammonia was also proportional to concentration. Under the experimental conditions, an amino acid concentration as low as 0.01 pmole could be determined without difficulty. Thus, the sensitivity of this reaction is higher than sensitivity of those reported previously. The optical densities of the color per 0.1 pmole of amino acid, at 570 rnp under the experimental conditions reported by Troll and Cannan (2)) Rosen (7), Fisher et al. (8), and Metheson and Tattrie (9) were around 0.002, 0.061, 0.037, and 0.21, respectively. By the present method, it was around 0.5, depending upon the individual amino acids (Fig. 4). Specificity of Color Development: Under our experimental conditions, urea reacted with ninhydrin so poorly compared with a-amino acids that it was not necessary to make any corrections (Fig. 4). A correction for the contribution by ammonia can be made by measuring ammonia concentration by Nessler’s reagent or the phenol-hypochloride method, or by drying samples after adding K,CO, to evaporate ammonia. Recommended Procedure: Add a sample containing 0.005-0.2 pmole of amino in 0.1 ml to 1.9 ml of a ninhydrin-citrate-glycerol mixture that consists of 0.5 ml of lo/O ninhydrin solution in 0.5M citrate buffer (pH 5.5), 1.2 ml of glycerol, and 0.2 ml of 0.5 M citrate buffer (pH 5.5), which were mixed in large quantities before the analysis. The final pH of the

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TAKAHASHI

FIG. 5. Amino acid concentration and color yield. The reaction mixture (2 ml), which consists of 0.35 M citrate, 5 mg ninhydrin, glycerol (60%), and tryptophan (0)) glycylglycine (O), NE+ (01, epinephrine (A), or tris (A), was heated in a boiling water bath for 10 min.

reaction mixture is 6.0. Heat in a boiling water bath for 12 min. Cool in a tap-water bath at room temperature. Shake the tube and read at 570 rnp within 1 hr. Run reagent blank and a standard amino acid solution at the same time to verify and standardize the determination. DISCUSSION

During studies of amino acid metabolism and protein biosynthesis, attempts were made to develop a simpler, more sensitive, and more specific assay for a-amino nitrogen for practical use. The present procedure can be said to have several advantages compared with those previously reported. Glycerol which is stable and nonvolatile, and has no odor, is a better medium for the color development. The reaction does not require other addtional compounds to facilitate the color yield. The reagents are stable even after mixing (the reagents which have been reported could not be mixed together before use because of instability). The present method does not require the dilution after the heating as described by Rosen (7). Color development is not affected by the common protein precipitants. The color is quite stable. The sensitivity is the highest so far reported. The reproducibility of the color development is

AMINO

ACID

DETERMINATION

77

good. Many analyses can be performed simultaneously and easily. There are two points which have not been improved: the color yield is not specific only for a-amino acids, and the color intensity is to some extent dependent upon the individual amino acids. SUMMARY

An improved calorimetric ninhydrin is described.

determination

of amino acids with the use of

ACKNOWLEDGMENTS This investigation was supported by a Research Grant AM 97985 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, and by the Hill Family Foundation. REFERENCES 1.

W. H., J. Biol. Chem. 136, 367 (1948). 2. TROLL, W., AND CANNAN, R. K., J. Biol. Chem. 200,803 (1953). 3. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 211, 907 (1954). 4. YEMM, E. W., AND COOKING, E. C., Analyst 80,209 (1955). 5. KALANT, H., Anal. Chem. 28,265 (1965). 6. YAMAGISHI, M., AND YOSHIDA, T., J. Pharm. Sot. (Japan) 73, 675 (1953). 7. ROSEN, H., Arch. Biochem. Biophys. 67, 10 (1957). 8. FISHER, L. J., BUNTING, S. L., AND ROSENBERG, L. E., C&n. Chem. 9, 573 (1963). 9. METHESON, A. T., AND TATTRIE, B. L., Can. J. Biochem. Ph&oZ. 42, 95 (1964). MOORE,

S., AND

STEIN,