THE QUANTITATIVE DETERMINATION OF PROLINE AND PIPECOLIC ACID WITH NINHYDRTN”

BY RICHARD S. SCHWEET

(From the Kerckhoff Laboratories of Biology, California Institute of Technology, Pasadena, California)

(Received for publication, November 25, 1953)

In recent years, a number of photometric methods for the quantitative determination of ar-amino acids have been introduced (1, 2). These meth- ods, based on reaction with ninhydrin in aqueous solution, are less satis- factory for the determination of imino acids.1 Grassmann and von Atnim (3) described reaction products of ninhydrin with imino acids and have suggested that these might be of use for the determination of proline. More recently, Chinard (4) has described a method which is applicable to proline, ornithine, lysine, and hydroxylysine. No quantitative determina- tion of the newly discovered pipecolic acid (5, 6) has been reported.

The present method is based on the formation of a red color when proline and pipecolic acid react with ninhydrin in glacial acetic acid under nearly anhydrous conditions. The absorption spectra of the colored products correspond with those isolated by Grassmann and von Arnim (3). Most primary amino acids, hydroxyproline, and ammonia give little or no color under these conditions. The sensitivity is similar to that of the ninhydrin methods used for or-amino acids (1, 2). In principle, this method is similar to the one described by Chinard (4). The conditions for color develop- ment used here are different and offer the advantages of greater sensitivity, speed of color development, and applicability to various types of mixtures including protein hydrolysates.

Early work yielded results which were variable owing to instability of the colored products. Attempts to stabilize the color by the addition of or- ganic solvents, such as ethanol, methyl Cellosolve, or pyridine, decreased the color yield. Reducing agents, such as stannous chloride (1) and as- corbic acid, gave color increases on a few occasions, but these were not re-

* These studies were supported (in part) by a grant from the Williams-Waterman Fund, a grant-in-aid to Dr. Henry Borsook from the American Cancer Society upon recommendation of the Committee on Growth of the National Research Council, and by a contract between the Office of Naval Research, Department of the Navy, and the California Institute of Technology, Division of Biology (No. NR-102-007). Part of this work was completed during the tenure of a postdoctoral fellowship from the National Institutes of Health, United States Public Health Service.

1 Although pipecolic acid gives a purple ninhydrin spot on paper, only a faint yellow color appears when color is developed by the usual ninhydrin methods (1, 2).

603

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604 NINHYDRIN DETERMINATION OF PROLINE

producible. The method of Troll and Cannan (2) for hydroxyproline produced very little color with proline, as they have noted. Pipecolic acid, also, cannot be determined by their method.

Color yields for proline were improved considerably by the addition of small amounts of hydrochloric or sulfuric acid, while pipecolic acid color formation was greatly inhibited. The basis of the effect of t,hese acids has not been studied. This observation has led to the development of meth- ods for the determination of each of the imino acids in the presence of the other. The procedure recommended permits the determination of these imino acids in aqueous solution, although the amounts of water present must be controlled. Duplicate determinations agree to within f2 per cent, and recoveries from mixtures average &5 per cent. The colored products formed by the methods finally adopted are not stable at high temperatures. However, the more rapidly the.color is formed, the higher the color yield. A short heating period at high temperature has thus given the best results (Fig. 1). Optimal color development also depends on the amount of ninhydrin used. This optimal amount for the pipecolic acid reaction and.for proline is more than 100 times the imino acid concen- tration. Troll and Cannan (2) have observed a similar phenomenon. The optimal ninhydrin concentration also depends on the amount of acid present in the case of proline. The final color formed decreases 3 to 5 per cent after an hour at room temperature. Reproducibility depends on ad- herence to the conditions of time and tempera%ure described below.

On the basis of these observations the methods described below were finally adopted and tested on primary amino acids, protein hidrolysates, and various nitrogenous compounds.

Reagents- 1. Ninhydrin solution. 372 mg. of ninhydrin are dissolved in 20 ml. of

glacial acetic acid. The solution should be freshly prepared each day. 2. Glacial acetic acid. Baker’s “analyzed reagent” in 1 pound bottles,

kept closed to prevent absorption of moisture, has been used in these de- terminations.

3. Hydrochloric-acetic acid mixture. 1 ml. of concentrated c.p. acid plus 99 ml, of glacial acetic acid.

4. Standard proline solution. 1 X 1F4 M proline (Merck and Com- pany) in glacial acetic acid. The solution appears to give the same color values for at least a month.

5. 3 M sodium nitrite solution. 20.7 gm. dissolved in 100 ml. of water. 6. 8.6 N hydrochloric acid. 10 ml. of concentrated c.p. acid plus 4 ml.

of water. 7. Ion exchange resin. Amberlite IR-4B (Rohm and Haas Company,

Philadelphia) is washed through two cycles of 2 N hydrochloric acid and sodium hydroxide. The last wash with sodium hydroxide is continued

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R. S. SCHXVEET 605

until the resin is chloride-free. Finally, the resin is washed with distilled water until the pH falls below 8.0.

General Method for Proline

Procedure-The analysis is applied to aliquots containing 1.5 to 10 y of proline in 18 X 150 mm. test-tubes. The water volume is adjusted to ex- actly 0.05 ml.23 3 0.2 ml. of the hydrochloric-acetic acid solution is added and glacial acetic acid to make a total volume of 3.85 ml. Finally, 0.3 ml. of ninhydrin reagent is added, and the tubes are covered with aluminum

0.6 r

1. o 5 IO 15 20 25 30

TIME (MIN.) FIG. 1. Effect of temperature on rate of proline color development.

given in the text; 0.05 FM of proline. Conditions

caps and placed in a glycerol bath at 121-122’. The level of the bath should be 3 inch above the liquid in the tubes. After exactly 5 minutes, the tubes are placed in an ice bath and cooled to room temperature. The solutions are adjusted to a convenient volume4 with glacial acetic acid, and the optical density at 530 rnp is determined within 15 minutes. Deter- minations are made in duplicate, and standards and reagent blanks are carried through the same procedure.

Results-The optical density curve (Fig. 2) follows Beer’s law within 2

2 Aqueous solutions of a volume greater than 0.05 ml. are taken to dryness at room temperature in a vacuum desiccator, and 0.05 ml. of water is added. In other cases dilutions are made with glacial acetic acid so that the aliquot used contains the correct water volume in a total volume which can be conveniently pipetted. When small water volumes must be pipetted, micro pipettes should be used (Micro- chemical Specialties Company, Berkeley, California).

3 The color yield of standards containing 0.1 ml. of water is 94 to 96 per cent. 4 The final volume for all the data reported here is 4.15 ml. Colors are read in the

Beckman spectrophotometer, 1 cm. cell.

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606 NINHYDRIN DETERMINATION OF PROLINE

per cent through readings up to an optical density of 0.8. There is a de- viation of 5 per cent from the straight line at an optical density of 1.2.

Although pipecolic acid also yields a similar color (Fig. 3), it is possible to determine proline in the presence of this imino acid by the method, since under these conditions the color yield from pipecolic acid is much less than that from proline (Table I). The optical density is read at, both 530 and 560 m/l, and the color due to pipecolic acid is subtracted. The contribu-

0.6 t

z gj 0.5

2 0.4 -

;: 0 k 0.3 - 0 0 PIPECOLIC ACID

$( , ‘:I’ , ,

0 0.01 0.02 0 03 0.04 0.05 006 0 07 0.08 IMINO ACID CONCENTRATION (J.lM’

FIG. 2. Effect of concentration on optical density. Each imino acid determined by the method given in the text.

tion of proline to the color at 530 rnp is calculated from the following equation,

DP,,o = 1.53 0530 - 0.93 Dw, (1)

where Dbso and D500 are the observed optical densities at these wave-lengths and DPBBo is the optical density due to proline at 530 rnp. Recoveries of proline (Table II) varied from 99.4 to 104.5 per cent by this method.

Since pipecolic acid has been found in appreciable quantities only in plants (5, 6), and probably is not normally a constituent of proteins (see the section on pipecolic acid), these calculations will be required only in special instances.

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R. S. SCHWEET 607

A number of other amino acids give small but significant color yields which would interfere with the proline determination under some condi- tions (Table I). The method has been applied by us mainly to metabolic studies of the imino acids in which these interferences appear to be neg- ligible. We have obtained good results by the method as described. However, the presence of salts may interfere with color development if present in sufficient concentration. Sodium chloride causes a decrease of 2 per cent in the color value when 1 to 2 PM is present per tube (20 to 40 times the proline concentration). Phosphate buffer is also inhibitory.

0’ 440 460 480 500 520 540 560 580 600 620

WAVELENGTH (MJI)

FIG. 3. Absorption spectra of proline and pipecolic acid compounds. Each imino acid determined by the method given in the text; 0.05 PM of each.

The above procedure was tested on protein hydrolysates with poor re- sults. It was noted that in the presence of amino acids at 10 times the concentration of imino acid the color yields from both proline and pipecolic acid were decreased. High concentrations of leucine, for example, pro- duced this effect (Table III). In order to remove these interferences, the following procedure, which is a modification of the method of Hamilton and Ortiz (7), was adopted.

Application to Protein Hyclrolysates-50 mg. of protein (or less if neces- sary) are hydrolyzed in a sealed tube with 4 ml. of 6 N hydrochloric acid for 18 hours at 105’. The insoluble humin is removed by centrifugation and washed with 1.0 ml. of water which is added to the hydrolysate. 0.3 ml. of sodium nitrite is mixed with 0.7 ml. of 8.6 N hydrochloric acid. When the reaction subsides, this is added to 1.0 ml. of protein hydrolysate and heated with shaking for exactly 2.5 minut,es in the bath at 122’. The

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608 NINHYDRIN DETERMINATION OF PROLINE

solution is cooled immediately, and essentially all of the strong anions are then removed by passage over the ion exchange resin, Amberlite IR4B.

The resin in the hydroxide form is pipetted onto a 0.9 X 10 cm. column5 and washed with distilled water until neutral. The volume of packed resin should be 0.9 X 6 cm. The solution after nitrous acid treatment is then quantitatively pipetted onto the column and the eluate collected in a 10 ml. volumetric flask. The flow rate is adjusted to about 0.1 ml. per minute. The column is washed three times with 2 ml. portions of water.

TABLE I

Color Yields of Amino Acids* The results are calculated for 0.1 PM of each amino acid. Proline and pipecolic

acid were determined at a concentration of 0.05 PM; other amino acids at 0.5 JAM.

Compound Proline method

Optical den- sity at 530 mp

Color yield, per cent

Proline............................. 1.04 100.0 Pipecolic acid. 0.150 14.4 Hydroxyproline 0.010 0.97 Ornithine 0.184 17.7 Citrulline. 0.173 16.6 Lysine 0.014 1.10 Cysteine. 0.065 6.25 Tryptophan. 0.009 0.087 Histidine........................... 0.002 0.19

Pipecolic acid method

Optical den- Color yield, sity at 560 mp per cent

0.208 19.3 1.10 100.0 0.013 1.18 0.044 4.00 0.038 3.46 0.050 4.55 0.033 3.00 0.012 1.09 0.003 0.27

* The following amino acids gave no color by either method: arginine, phenyl- alanine, tyrosine, glycine, methionine, aminoadipic acid, alanine, valine, leucine, glutamic acid, aspartic acid, serine, threonine, and cystine. Other compounds tested, all of which yielded no color, were glutathione, glutamine, allantoin, creatine, glycine anhydride, and uracil.

The eluate and washings are collected and made to volume. The final pH should be 4 or higher.

When proteins containing 10 to 15 per cent proline are being studied, the final eluate contains 100 to 150 y of proline per ml. Since the best range for the determination is 3 to 8 y per tube, further dilution is required. For convenient pipetting, the solution is diluted to 25 to 40 y of proline per ml. with glacial acetic acid, and 0.1 and 0.2 ml. aliquots are determined by the usual procedure. The appropriate amount of water to bring the water volume to 0.05 ml. in all the tubes is added. When the proline con-

5 These are conveniently prepared by drawing out the end of a 1 cm. glass tubing to 4 mm. inner diameter. Glass wool is packed into the bottom, and the flow rate is controlled with a small piece of plastic tubing and screw clamp fastened to the end. The hold-up volume of the resin column is 3.0 ml.

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R. s. ~CI~WEET 609

tent of the original protein is low, it may be necessary to concentrate the aliquots of the final eluate to dryness in a vacuum desiccator before the determination is made. The usual 0.05 ml. of water is then added and the analysis carried through as described. Standards dried in this way showed no loss.

Results-To test the method, a mixture of proline and leucine was taken through the procedure described above. Recoveries averaged 96.0 per cent (Table III). Recoveries from duplicate runs agreed to within ~t2 per cent. Several proteins were then analyzed by this procedure. The percentage proline found (gm. of proline per 100 gm. of dry, ash-free protein) was as follows: gelatin6 14.05, casein 11.0, zein 10.60, and gliadin

TABLE II

Recoveries of Imino Acids from Mixtures

Each tube contained the indicated amounts of proline or pipecolic acid plus 0.04 PM of the other imino acid. Determinations and calculations were carried out according to the procedure for the respective imino acid.

Proline Imino acid added

DP at 530 m# Recoveryt

PM per cent

0 0.0 0.01 0.115 104.5 0.02 0.220 102.2 0.04 0.432 100.9 0.08 0.835 99.4

* See Equation 1. t Calculated from the data of Fig. 2. $ See Equation 2.

Pipecolic acid

DP at 560 rnpt Recoveryt

per cent

0.003 0.109 99.1 0.211 96.4 0.433 98.4 0.838 96.3

12.90. Tristram (8) has reported 14.8, 11.6 (average), 10.53, and 13.35 per cent proline, respectively, for these proteins. The values are slightly lower, therefore, than those reported in the literature, but have not been corrected for the proline loss during nitrous acid treatment (Table III). This correction would be valid since it is reproducible. Hamilton and Ortiz (7) have noted errors resulting from the nitrous acid treatment of ornithine, arginine, and citrulline. The color yield from lysine and ar- ginine by the method described here was 1.06 and 2.1 per cent of proline, respectively. The method is not applicable in the presence of ornithine,

6 These proteins were obtained from commercial sources and were not further purified. The following proteins were used: gelatin, Knox sparkling; casein, vita- min-free, Labco; aein, Corn Products Refining Company; gliadin, Bios Laboratories. Casein and zein contained 15.4 and 15.0 per cent nitrogen, respectively, according to the manufacturer.

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610 NINHYDRIN DETERMINATION OF PROLINE

since the nitrous acid treatment converts it to proline (9). If present, ornithine would have to be removed. Ion exchange chromatography with IRC-50 has been suggested for a similar purpose (10). Citrulline, if present, would also have to be removed, and this is not readily accom- plished by ion exchange.

It is concluded from these studies that the proposed method may be ap- plied without modification in many instances. The sensitivity of color development to interference may require preliminary separation at times. Proteins may be analyzed by the procedure described, provided corrections for proline losses are made and the small positive errors associated with the particular protein being analyzed are taken into account.

TABLE III

Recoveries of Imino Acids from Mixtures with Leucine

Each tube contained the indicated amount of proline or pipecolic acid plus 10 times that amount of leucine.

Imino acid added

lrdd

0.020 0.025 0.040 0.050 0.080

Proline recovery* Pipecolic acid recovery*

Untreated Treated with nitrous acidt

per cent

85.0

82.6 89.5

87.2 92.4

per cent

89.2

per cent

96.0 96.5

95.9 94.3

Untreated Treated with nitrous acidt

* Calculated from the data of Fig. 2. t 1 ml. of 6 N hydrochloric acid containing 10 PM of imino acid plus 100 PM of leu-

tine treated with nitrous acid according to the procedure for protein hydrolysates. The pipecolic acid was also dried as described in the text.

General Method for Pipecolic Acid

Procedure-The analysis is applied to aliquots containing 1.5 to 10 y of pipecolic acid’ in 13 X 150 mm. test-tubes. The water volume is ad- justed to exactly 0.05 ml.29 * Glacial acetic acid is added to make a total volume of 3.95 ml. Finally, 0.2 ml. of ninhydrin reagent is added, and the tubes are covered with aluminum caps and placed in a water bath as de- scribed for the proline determination. The tubes are heated for 8 minutes and then placed in an ice bath and cooled to room temperature. The solu- tions are adjusted to a convenient volume* with glacial acetic acid, and the

7 nn-Pipecolic acid, a gift from Dr. P. Lowy, was used for the determination of standards. This was an analytically pure sample.

8 The color yield of standards containing 0.1 ml. of water is 88 to 90 per cent.

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IL S. SCHWEET 611

optical density at 560 rnp is determined within 15 minutes. Determina- tions are made in duplicate, and standards and reagent blanks are carried through the same procedure.

Results-The optical density curve by this procedure is similar to that of proline (Fig. 2). The yield of proline color is much less than that from pipecolic acid in the absence of hydrochloric acid (Table I); hence it is possible to determine pipecolic acid in the presence of proline by reading the optical densities at both 530 and 560 rnp and correcting for the color due to proline. The pipecolic acid contribution to the optical density is calculated from the following equation:

DP66o = 1.48 0660 - 0.76 Dam (2)

where 0530 and D560 are the observed optical densities at the respective wave-lengths and DP660 is the optical density due to pipecolic acid at this wave-length. Determinations of mixtures of the two imino acids by this procedure gave recoveries ranging from 96.3 to 99.1 per cent (Table II). Color yields of 3 to 4 per cent of pipecolic acid (Table I) are obtained from the dibasic amino acids.

Pipecolic acid may be determined in the presence of large amounts of a-amino acids by the procedure given for the determination of proline in protein hydrolysates in the preceding section. Recoveries from mixtures with leucine were 94.3 to 96.5 per cent by this procedure (Table III). Lysine and arginine when treated by the nitrous acid procedure gave color yields of 3.1 and 0.91 per cent, respectively. The nitrous acid treatment of lysine appears to yield a small amount of pipecolic acid, based on the absorption spectrum (after ninhydrin treatment) and paper chromatog- raphy.

Color development from pipecolic acid is inhibited by small amounts of hydrochloric acid. A convenient procedure for the removal of volatile acid is to pipette aliquots of the eluate (or dilutions) into the test-tubes which are to be used for color development. These are placed in a vacuum desiccator over sodium hydroxide and taken to dryness by means of a vacuum pump and a dry ice trap. 0.2 ml. of water is added, and the tubes are again dried. This is repeated a third time. The tubes are then re- moved, 0.05 ml. of water is added, and the usual procedure is followed.

Pipecolic acid was determined on the four protein hydrolysates used for proline analysis and the mixed proteins of a Neurospora mutant. Equa- tion 2 was used to calculate the pipecolic acid content with optical density readings at 530 and 560 rnp. The color values were then corrected for color formation from lysine. The values show that no pipecolic acid was present. It is estimated that, because of the large amount of proline present, these determinations indicate that the pipecolic acid content is

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612 NINHYDRIN DETERMINATION OF PROLINE

less than 0.5 per cent. However, paper chromatography of these hydrol- ysates and the protein from a Neurospora mutant (which formed pipecolic acid from lysine in the non-protein nitrogen fraction) has shown that 0.2 per cent pipecolic acid or less is present in these proteins.9

Products of Reaction

On the basis of the absorption spectra (Fig. 3) and the conditions under which the colors are formed, it is probable that the colored compounds are the di(diketohydrindylidene)-pyrrolidine and -piperidine compounds iso- lated by Grassmann and von Arnim (3). These authors reported absorp- tion maxima at 528 and 554 rnp, respectively, for the two compounds. Under the conditions described here the peaks are at 535 and 558 rnp. We have not attempted to isolate these compounds.

In addition, we have observed that all of the a-amino acids form transi- ent colors during the heating period. Only the basic amino acid and cysteine colors remain, however. The absorption spectra of these colored compounds differ from those of the imino acids and are different for each of the amino acids, and thus they are not diketohydrindylidene-diketo- hydrindamine, the product of the reaction with a-amino acids in aqueous solution (1, 2). Baikiain’O (A4-dehydropipecolic acid) gives a color yield of 65 per cent of pipecolic acid, with an absorption maximum at 400 mp.

SUMMARY

1. The reaction of proline and pipecolic acid with ninhydrin in glacial acetic acid in the presence and absence of hydrochloric acid is used as the basis for a quantitative, photometric determination for each of the imino acids.

2. Procedures are described for the application of this method to the analysis of mixtures of the two imino acids and mixtures with a-amino acids, including protein hydrolysates.

3. No evidence has been found for the presence of pipecolic acid in any of five proteins studied.

The author wishes to express thanks to Dr. Henry Borsook for his en- couragement and support of this work. The competent technical as- sistance of Mr. R. J. Busser is acknowledged.

BIBLIOGRAPHY

1. Moore, S., and Stein, W. H., J. Biol. Chem., 176, 367 (1948). 2. Troll, W., and Cannan, R. K., J. Biol. Chem., 200, 803 (1953).

Q Schweet, R. S., Holden, J. T., and Lowy, P., in preparation 10 A gift from Dr. F. E. King.

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R. S. SCHWEET 613

3. Grassmann, W., and van Amim, K., Ann. Chem., 509, 288 (1934). 4. Chinard, F. P., J. Biol. Chem., 199, 91 (1952). 5. Morrison, R. I., Biochem. J., 53, 474 (1953). 6. Zacharius, R. M., Thompson, J. F., and Steward, F. C., J. Am. Chem. Sot., 74,

2949 (1952). 7. Hamilton, P. B., and Ortiz, P. J., J. Biol. Chem., 187, 733 (1950). 8. Tristram, G. R., Advances in Protein Chem., 5, 83 (1949). 9. Hamilton, P. B., J. Biol. Chem., 198, 587 (1952).

10. Block, R. J., and Bolling, D., The amino acid composition of proteins and foods, Springfield, 2nd edition, 434 (1951).

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Richard S. SchweetPIPECOLIC ACID WITH NINHYDRINDETERMINATION OF PROLINE AND

THE QUANTITATIVE

1954, 208:603-614.J. Biol. Chem. 

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