PROLINE AND IIYDROXYPROLINE: PURIFICATION, RE- ACTION WITH NINHYDRIN, AND SOME PROPERTIES OF THEIR N-NITROSO DERIVATIVES

PROLINE AND IIYDROXYPROLINE: PURIFICATION, RE- ACTION WITH NINHYDRIN, AND SOME PROPERTIES OF THEIR N-NITROSO DERIVATIVES BY PAUL B. HAMILTON AND PRISCILLA J. ORTIZ (From the Alfred I. du Pont Institute of The Nemours Foundation, Wilmington, Delaware) (Received for publication, December 19, 1959) To standardize a new method developed in this laboratory for the determination of the imino carboxyl nitrogen2 of proline and hydroxyproline in the products of protein hydrolysates, without preliminary removal of amino or non-imino nitrogen, pure proline and hydroxyproline were necessary. All commercial samples tested were contaminated by measurable amounts of substances capable of evolving nitrogen gas in the Van Slyke nitrous acid procedure (2). Because determination of proline and hydroxyproline imino carboxyl nitrogen by the ninhydrin-co2 technique (3) forms an integral part of the new method, and also because proline evolves CO2 in excess of 1 mole per mole of imino acid on heating at 100 in the ninhydrin reaction for periods longer than 3.2 minutes (4), a reexamination of the reaction of both imino acids with ninhydrin (3, 4) was undertaken. Preliminary treatment of amino acid mixtures with nitrous acid to convert all amino acids except proline and hydroxyproline to their respective cr-hydroxy derivatives is employed in the method referred to above. While investigating the proper use of nitrous acid in this preliminary step, we discovered conditions for the quantitative conversion of proline and hydroxyproline to their respective N-nitroso derivatives, and some of the properties of these derivatives were investigated. Analytical Procedure a-amino or or-lmino Carboxyl Nitrogen-Carboxyl nitrogen was determined by the method of Van Slyke, Dillon, MacFayden, and Hamilton (3). The reagents used in the manometric procedure were nearly saturated with NaCl, as recommended by MacFadyen (5), and aldehyde errors were minimized by the use of hydrasine, as suggested by Schott, 1 Hamilton, P. B., and Ortiz, P. J., unpublished work. z In conformity with the nomenclature of previous publications (Hamilton (1) p. 375 foot-note), the term imino carboxyl nitrogen is used to indicate values calculated as 1 gm. atom of nitrogen per mole of COZ evolved by reaction of the &nino acids, proline and hydroxyproline, with ninhydrin. 607

608 PROLINE AND HYDROXYPROLINE Rockland, and Dunn (4), but with lactic acid solutions containing hydrazine (6). Ninhydrin was crystallized several time$ from 2 N HCl until 100 mg. of the purified reagent evolved no COZ on being heated for 20 minutes in a blank analysis at ph 2.5. Octyl alcohol, used as an antifoaming agent, was distilled to remove substances capable of contributing COZ and tested in a blank analysis. Black alundum grains (No. 14 mesh), used to promote smooth boiling, contain ferrous iron which renders them unsuitable. White alundum grains were satisfactory after having been boiled for 2 hours in 6 N HCl, rinsed thoroughly with distilled water, and air-dried. Reactions were carried out at 100 in all-glass vessels (Hamilton and Van Slyke (7)) on 5 ml. volumes of solution with 100 mg. of ninhydrin at the ph values and for the times of heating mentioned in the experimental part. Unless otherwise specified, blank analyses were carried out with all the reagents included, and all analyses were made in duplicate. Amino Nitrogen (Nitrous Acid Nitrogen)-Amino nitrogen was determined by the Van Slyke nitrous acid procedure (2) with a reaction time of 5 minutes. Total (KjeZduhZ) Nitrogen-Total nitrogen was determined by the macro- Kjeldahl procedure of Hiller, Plazin; and Van Slyke (8) with mercury as the catalyst and a digestion time of 6 hours. EXPERIMENTAL PuriJication and Properties of Proline and Hydroxyproline Putijkation of Proline by Rhodanilate-Purification of n-proline was first attempted with rhodanilic acid, as recommended by Bergmann (9). The proline isolated was always slightly colored, was markedly hygroscopic, and developed a strong orange-yellow color when dissolved in warm ethanol, properties which are not those of freshly prepared pure proline and which apparently arise from contamination with traces of rhodanilic acid. Repeated crystallization reduced the yield to 50 per cent of the starting material without a satisfactory product having been obtained. Purification by means of the picrate was accordingly investigated. PurQication of Proline As Picrate-To proline dissolved in approximately 15 times its weight of glacial acetic acid, picric acid was added in slight excess over 1 mole, as recommended by Alexandroff (10) ; the solution was completed by warming. On adding 30 volumes of anhydrous diethyl ether, proline picrate rapidly separated; crystallization was completed in 2 to 3 hours at 4. The picrate was dried in vacua for 16 hours over calcium chloride (94 per cent). Four crystallizations were 8 Hamilton, P. B., and Ortis, P. J., And. Chem., in press.

P. B. HAMILTON AND P. J. ORTIZ 609 often necessary to eliminate traces of substances capable of evolving nitrogen gas in the Van Slyke nitrous acid procedure and to obtain a product which gave theoretical yields of carboxyl nitrogen and had a melting point of 154 (corrected). These properties were unchanged by a fifth crystallization. With each recrystallization, picric acid was added to provide approximately 0.005 mole excess. Otherwise dark brown crystals tended to form, and the amount of these increased with each subsequent crystallization. The picrate was suspended in water, decomposed by means of aniline, and the free proline, isolated according to the method of Cox and King (ll), was recrystallized from warm ethanol on the addition of an equal volume of anhydrous diethyl ether. In a typical experiment, the product of the fourth crystallization contained less than 0.05 per cent of its total nitrogen as amino nitrogen, gave a carboxyl nitrogen of 1.00 gm. atom per mole, and melted at 220.4 (corrected). A fifth crystallization did not alter these values. Analysis- Calculated. C 52.16, H 7.88, N (Dumas) 12.17 Found. 51.26, 7.33, 12.02, ash 0.17 Freshly prepared proline was odorless, but within half an hour a characteristic musty smell was discernible. Fresh preparations of pure proline were not hygroscopic, since 100 mg., exposed for 3 weeks, showed no detectable change in weight. However, on prolonged exposure to air and light, it became hygroscopic and noticeably pink or brown in color. In an evacuated sealed glass ampul protected from light, it neither caked nor became discolored after 8 months storage. In general, the more crude the preparation, the more rapidly these changes took place. These findings appear to reconcile the observation of Towne (12) that his preparations were non-hygroscopic and that of Bergmann (9), who stated that his preparations were hygroscopic. Hydroxyproline could be purified similarly by means of picric acid, but the yields were approximately 20 per cent lower because of the greater solubility of hydroxyproline picrate as compared with proline picrate in glacial acetic acid-ether mixtures. Pure hydroxyproline remained odorless and non-hygroscopic, but, when kept in loosely stoppered test-tubes, became noticeably pink in 8 months time, whether in light or in dark. No change was noted in samples placed in sealed evacuated ampuls and stored either in light or in darkness. 4 The authors wish to thank Dr. A. Elek of the Elek Micro Analytical Laboratories, 4763 West Adams Boulevard, Los Angeles 16, California, for the elementary anjyse4.

610 PROLINE AND HYDROXYPROLINE Recently, satisfactorily pure n-proline and hydroxy-n-proline have become available commercially.5 Reaction of Proline and Hydroxyproline with Ninhydrin Proline or hydroxyproline was dissolved in water or hydrochloric acid solution adjusted to ph 1. For analysis at ph 2.5 or 4.7, 100 mg. of appropriate solid citrate buffer (3) were added to 5 ml. of the water solution. For analysis at ph 1, 5 ml. of the solution were employed with no buffer II I 4 5 6 7 8 9 IO II 12 I3 I4 I5 I6 I7 I8 I9 20 TIME OF HEATING AT 100 C- MINUTES FIG. 1. Proline carboxyl nitrogen as a function of ph and time of heating at 100. All reactions were carried out in triplicate on 5 ml. volumes of solution with 100 mg. of ninhydrin. added. The vessels were charged with 100 mg. of ninhydrin and heated at 100 for the time indicated in Fig. 1. Upon interpolation from Fig. 1, calculated theoretical carboxyl nitrogen values for proline were obtained on heating the vessels for 4 minutes at ph 4.7, 6 minutes at ph 2.5, and 20 minutes at ph 1.0. The apparent discrepancy between the 3.2 minutes heating at ph 2.5 found necessary by Schott, Rockland, and Dunn (4) to obtain theoretical carboxyl nitrogen values and the 6.0 minutes heating at the same ph reported in this paper resides in the fact that the former authors employed 50 mg. of ninhydrin per ml. of amino acid solution analyzed, whereas only 20 mg. of ninhydrin 6 H. M. Chemical Company, 144 North Hayworth Avenue, Hollywood, California.

P. B. HAMILTON AND P. J. ORTIZ 611 per ml. were employed in the present work. Both experiments are correctly reported ((3) p. 643). Heating for 6 minutes at 100 at ph 4.7 evolved a greater excess of COZ than at ph 2.5, whereas, if heating was prolonged to 20 minutes at 109, the excess CO2 evolved was greater at ph 2.5 than at ph 4.7. No explanation can be offered for this effect. Hydroxyproline values are not shown, since COZ in excess of the calculated theoretical of 1 mole per mole of imino acid was never evolved. N-Nitrosoproline and N-Nitrosohydroxyproline As originally found by Van Slyke (2), neither imino acid yields nitrogen gas in the nitrous acid procedure, but a reaction does take place, since a product was readily extracted by means of diethyl ether after acidification to ph 1. Evaporation of the ether left a non-crystalline residue..this residue dissolved readily in 6 N HCl and, after 90 minutes boiling followed by evaporation, a white crystalline solid was obtained. Two recrystallizations from alcohol-ether yielded pure proline. By starting with 5 gm. of proline, 4.4 g-m. (86 per cent) were recovered. These findings are compatible with the following equations: (1) R=NH+HONO ph 2.5 R=N-NO+HzO -+ (2) R=N-NO+HzO 6~Hcl R=NH+HONO Reasonably satisfactory evidence of the position of the NO group (Equation 1). was provided by the observation that the N-nitroso derivatives of proline or of hydroxyproline do not react with ninhydrin in the ninhydrin-coz method. This would be probable only if the hydrogen of the imino nitrogen atom were replaced by an NO group. Reaction of a-amino acids with ninhydrin has been shown to be abolished ((3) p. 630) upon complete substitution of the nitrogen. By using the reaction of Bratton and Marshall (13) for the detection of aryl amines, the formation of nitrous acid from N-nitrosoproline (Equation 2) was demonstrated. To a solution of N-nitrosoproline, solid ammonium sulfamate was added to decompose free nitrous acid, until frothing ceased on further additions. The solution was cooled and a few crystals of p-aminobenxoic acid and 1 mg. (knife point) of N-(l-naphthyl)-ethylenediamine were added; the solution was then acidified with 2 ml. of concentrated HCl. No color formed, indicating absence of free nitrous acid, but, on being gently warmed, the solution rapidly developed an intense purple color. Nitrous acid produced by acid hydrolysis of the N- nitrosoproline (Equation 2) reacted to form the diazotized aryl amine which combined with the coupling agent to form an azo dye.

612 PROLINE AND HYDROXYPROLINE This result supports the postulate of Hiller and Van Slyke (14) that proline and hydroxyproline exhibit reactions with nitrous acid that are typical and characteristic of secondary amines. Formation of N-Nitrosoproline and N-Nitrosoh@oxyproline-To prepare a solution of N-nitrosoproline, 50 mg. of proline were dissolved in 75 ml. of 0.3 N HCl, 3 gm. of solid NaNOz were added, and the flask was immersed in a boiling water bath (100 ) for 5 minutes. The flask was then cooled and the solution diluted to 100 ml. with water. N-Nitrosohydroxyproline and blank solutions were prepared similarly. Carboxyl Nitrogen of N-Nitrosoproline and N-Nitrosohydroxyprok---A 2 ml. portion of N-nitrosoproline solution and 2 ml. of water were placed in an all-glass reaction vessel, and 1 ml. of 60 per cent ammonium sulfamate solution (LaMotte, pure standard) was added slowly with cooling. With bromocresol green (0.04 per cent, aqueous) as an internal indicator, the reaction was adjusted to yellow-green by the dropwise addition of 5 N NaOH, followed by 1 N HCl. The solution was adjusted exactly to ph 2.5 by t,he addition of 100 mg. of solid citrate buffer (3) and analyzed for carboxyl nitrogen, with 100 mg. of ninhydrin and a heating time of 6 minutes. N-Nitrosohydroxyproline and blank solutions were analyzed similarly. The N-nitroso derivatives of proline and hydroxyproline gave respectively 0.01 and 0.02 atoms of carboxyl nitrogen per mole, indicating that conversion of each imino acid to its respective N-nitroso derivative was at least 98 to 99 per cent complete. Hydrolysis of N-Nitrosoimino AtideA 5 ml. portion of N-nitrosoproline solution, 1.2 gm. of solid ammonium sulfamate, and 15 ml. of 8 N HCl were placed in a 100 ml. Kjeldahl flask and boiled vigorously. 6 N HCl was added as necessary, but fluid was allowed to boil away until 4 to 5 ml. remained after 90 minutes. N-Nitrosohydroxyproline and blank solutions were prepared similarly. The procedure effected the following reactions: N-Nitrosoproline was hydrolyzed to yield free proline and nitrous acid; nitric acid was reduced to nitrous acid; part of the nitrous acid reacted with ammonium sulfamate and part decomposed to oxides of nitrogen which escaped; ammonium sulfamate was hydrolyzed to ammonium sulfate. The residual 4 to 5 ml. of hydrochloric acid were distilled off in vacua, the dry residue was taken up in 5 ml. of water, again evaporated, and finally dissolved in 2 ml. of water. This solution was adjusted to ph 2.5 (approximately) and made up to 11.0 ml. with water. Carboxyl nitrogen was determined as described above. Carboxyl nitrogens of the hydrolyzed N-nitrosoproline and N-nitrosohydroxyproline were found to be respectively 0.98 and 0.97 atoms per

P. B. HAMILTON AND P. J. ORTIZ 613 mole, indicating practically quantitative reconversion of the N-nitroso derivatives to the free imino acids. Extraction of N-Nitrosoimino Acids by Means of Ether-A 5 ml. sample of the N-nitrosoproline solution, 1 ml. of 60 per cent ammonium sulfamate, and 1 ml. of 6 M phosphoric acid were placed in a glass thimble and extracted with ether in a Soxhlet apparatus as described by Gould (15). The receiving flask was charged with 10 ml. of 6 N HCl and 250 ml. of diethyl ether. N-Nitrosoproline solution was extracted for 4 hours, the N-nitrosohydroxyproline solution for 16 hours. After extraction, ether was evaporated and the residual acid solution in the receiving flask transferred to a Kjeldahl flash with 6 N HCI. Hydrolysis, removal of excess acid, neutralization, and analysis for carboxyl nitrogen were carried out as described previously. Recovery of proline and hydroxyproline carboxyl nitrogen were 0.98 and 0.97 atoms per mole respectively. The carboxyl nitrogen lost could not be demonstrated in the extracted residue in the thimble. Apparently, destruction of small amounts of imino acids took place. This is not unexpected, as solutions of N-nitrosoproline or N-nitrosohydroxyproline containing excess nitrous acid lose 52 and 39 per cent respectively of their original carboxyl nitrogen on standing for 4 weeks at room temperature. Extraction of N-Nitrosoproline and N-Nitrosohydroxyproline As Function of ph-above ph 4.0, neither N-nitrosoproline nor N-nitrosohydroxyproline could be extracted from an aqueous solution by ether. At lower ph values, the N-nitroso derivatives were extracted, the amount becoming maximal at approximately ph 1. Extraction of N-Nitrosoproline from Acid Aqueous h olution by Organic Solvents-10 ml. portions of a solution of N-nitrosoproline acidified to ph 1 were shaken with 60 ml. portions of methyl acetate, ethyl acetate, n-propyl acetate, isopropyl acetate, methylethyl ketone, or methylal. 50 ml. of solvent were removed and proline carboxyl nitrogen was determined. These solvents extracted 1.5 to 1.7 times as much N-nitrosoproline as diethyl ether. In a similar experiment, only a third as much N-nitrosohydroxyproline as N-nitrosoproline was extracted by ether. The ability of solvents other than ether to extract N-nitrosohydroxyproline was not investigated. Diethyl ether was considered the solvent of choice because of its low boiling point and ease of removal, and because it contributes nothing to a blank analysis. DISCUSSION The discrepancy in statements in the literature regarding hygroscopic properties of proline appears to be reconciled by the observations described. Preparations of proline can apparentlv be kent unaltered for

614 PROLINE AND HYDROXYPROLINE only a relatively short time when exposed to light and air. Proline is pure white and is not hygroscopic at first but may become hygroscopic and discolored in time. Storage in evacuated sealed glass ampuls protected from light seems to be the most satisfactory way of preventing change. The finding of Schott, Rockland, and Dunn (4) that proline evolves COz in excess of 1 mole per mole of imino acid on continued heating in the ninhydrin analysis is confirmed. Hydroxyproline, on the other hand, does not evolve more than 1 mole of CO2 per mole of imino acid. Application of the principles described makes possible quantitative methods for the determination of total imino carboxyl nitrogen in protein hydrolysates and these will be described in a subsequent publication. Also, since proline or hydroxyproline carboxyl nitrogen can be determined after reaction with nitrous acid, either in the reaction mixture or after extraction of the N-nitroso derivative by ether, procedures are provided whereby purity of these imino acids may be checked. A single procedure can therefore be substituted for separate determinations of carboxyl nitrogen (3) and amino nitrogen (2). For conditions outlined in previous sections of this paper, proline. or hydroxyproline nitrogen values are multiplied by 1.02 or 1.03 respectively to correct for loss of carboxyl nitrogen. These losses are sufficiently constant and reproducible so that, for pure imino acids, corrected values are within fl per cent of theoretical. A single attempt to isolate proline and hydroxyproline from a hydrochloric acid hydrolysate of gelatin by means of ether extraction of the N-nitroso derivatives gave low yields, as the experimental conditions selected were not ideal. The authors gratefully acknowledge the interest and encouragement of Dr. D. D. Van Slyke, Hospital of The Rockefeller Institute for Medical Research, New York, in whose laboratory this work had its origin, and of Dr. Lee E. Farr, Director of Research, Alfred I. du Pont Institute of The Nemours Foundation, under whom it has been continued. SUMMARY 1. Purification of proline or hydroxyproline by means of the picrate is described. Proline is a white, crystalline, non-hygroscopic solid with a characteristic odor when freshly prepared. Unless stored with protection from air and light, it becomes caked, discolored, and markedly hygroscopic in time. Hydroxyproline discolors but does not become hygroscopic. 2. The finding of Schott, Rockland, and Dunn (4) that proline evolves

P. 8. HAM~LTOE AND P. J. ORTIZ 615 carboxyl nitrogen in excess of 1 gm. atom per mole in the ninhydrin reaction is confirmed. Theoretical yields of CO2 are evolved from proline after 6 minutes at 100 with 20 mg. of ninhydrin per ml. at ph 2.5 in the ninhydrin method (3). At ph 4.7 and the same concentration of ninhydrin, theoretical yields of CO2 are obtained after 4 minutes at 100. 3. Proline and hydroxyproline, when heated at 100 in solutions of approximately ph 2.5 containing nitrous acid, are converted quantitatively to their respective N-nitroso derivatives. Hydrolysis of the N- nitroso derivatives yields 98 and 97 per cent respectively of the original acid, as measured by the recovery of imino carboxyl nitrogen. 4. N-Nitrosoproline and N-nitrosohydroxyproline can be extracted from acid aqueous solutions (below ph 4.0) by various organic solvents. The conditions are outlined whereby 98 and 97 per cent respectively of each imino acid can be recovered by extraction with ether. 5. Procedures taking advantage of the properties of proline and hydroxyproline and their N-nitroso derivatives are indicated whereby these imino acids may be determined in acid protein hydrolysates, their purity may be checked, and they may be isolated from protein hydrolysates. BIBLIOGRAPHY 1. Hamilton, P. B., J. Biol. Chem., 168, 375 (1945). 2. Van Slyke, D. D., J. Biol. Chem., 83, 425 (1929). 3. Van Slyke, D. D., Dillon, R. T., MacFadyen, D. A., and Hamilton, P., J. Biol. Chem., 141, 627 (1941). 4. Schott, H. F., Rockland, I,. B., and Dunn, M. S., J. BioZ. Chem., 164,397 (1944). 5. MacFadyen, D. A.,.I. Biol. Chem., 146, 387 (1942). 6. Hamilton, P. B., and Van Slyke, D. D., J. Biol. Chem., 160, 231 (1943). 7. Hamilton, P. B., and Van Slyke, D. D., J. Biol. Chew, 184, 249 (1946). 8. Hiller, A., Plazin, J., and Van Slyke, D. D., J. BioZ. Chem., 176, 1401 (1948). 9. Bergmann, M., J. BioZ. Chem., 110, 471 (1936). 10. Alexandroff, D., 2. physiol. Chem., 46, 17 (1995). 11. Cox, G. J., and King, H., J. BioZ. Chem., 84, 533 (1929). 12. Towne, B. W., Biochem..I., 30, 1837 (1936). 13. Bratton, A. C., and Marshall, E. K., Jr., J. BioZ. Chem., 128, 537 (1939). 14. Killer, A., and Van Slyke, D. D., J. BioZ. Chem., 39, 479 (1919). 15. Gould, B. S., Science, 98, 546 (1943).

PROLINE AND HYDROXYPROLINE: PURIFICATION, REACTION WITH NINHYDRIN, AND SOME PROPERTIES OF THEIR N-NITROSO DERIVATIVES Paul B. Hamilton and Priscilla J. Ortiz J. Biol. Chem. 1950, 184:607-616. Access the most updated version of this article at http://www.jbc.org/content/184/2/607.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/184/2/607.citation.full.h tml#ref-list-1