THE COMBINED AMINO ACIDS IN SEVERAL SPECIES OF MARINE ALGAE*

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1 THE COMBINED AMINO ACIDS IN SEVERAL SPECIES OF MARINE ALGAE* BY DONALD G. SMITH AND E. GORDON YOUNG (From the Maritime Regional Laboratory, National Research Council, Halifax, Canada) (Received for publication, May 17, 1955) The distribution of the free and combined amino acids in a few algae has recently been reported (1,4, 7-12, 23, 25). These analyses have been both qualitative and quantitative and are mostly fragmentary with the exception of those for Fucus vesiculosus (25) and for Chlorella vulgaris and a few other unicellular green algae (12). Those for ChZoreZZa have been widely divergent (9, 11, 23). Our earlier work on Fucus has therefore been extended to include a study of the combined amino acids of four other common seaweeds, Ascophyllum nodosum, Chondrus crispus, Rhodymenia palmata, and Ulva Zactuca, as representative species of the three major classes of algae. EXPERIMENTAL Methods-Specimens were collected in bulk locally, dried in a forced draft of warm air, and powdered in a Wiley mill. Free amino acids and simple peptides were extracted by shaking the dried, ground weed with 75 per cent ethanol for 24 hours, in the proportion of 1 gm. to 100 ml. of solvent. The residue was boiled in 20 per cent hydrochloric acid for 24 hours, in the proportion of 1 gm. of dry material to 200 ml. of acid. The insoluble humin was filtered quantitatively. Excess acid was removed from the filtrate by repeated distillation in VUJW). The residue was taken up in a volume of water to give a final concentration of approximately 1 mg. of N per ml. The solution was neutralized with silver oxide until a ph of about 4.5 was established. It was then centrifuged at 5000 r.p.m. until clear. The precipitate of silver chloride may have contained some cystine and any purines present, but no appreciable loss of nitrogen was ever detected in this procedure. Nitrogen was estimated by the micro-kjeldahl procedure. The amino acids formed on hydrolysis were determined quantitatively by the technique of ion exchange of Moore and Stein (21). They were also identified by the descending technique of paper chromatography. The papers were treated with borate buffer of ph 9 prior to one-dimensional chromatography (20). The solvent was phenol equilibrated against the * Issued as Publication No of the National Research Council. 845

2 846 AMINO ACIDS OF MARINE ALGAE same borate buffer. The procedure of Levy and Chung (16) was employed for two-dimensional chromatograms. The color reagents were 0.2 per cent ninhydrin in 95 per cent ethanol containing about 20 per cent by volume of acetic acid and 1 per cent p-dimethylaminobenzaldehyde in 1 N hydrochloric acid (10). Results The time of collection and the forms of nitrogen determined are listed in Table I. In agreement with the more detailed results for Fucus (25), the nitrogen in the other four species was about 60 to 70 per cent as protein nitrogen and 10 to 33 per cent in other forms, such as free amino acids, peptides, organic bases, pukes, pyrimidinea, etc. The lower values for nitrogenous material soluble in ethanol in Rhodymmia and Ulva may have Species F. vesiculosus A. nodosum c. crispus R. palmata U. lactuca - _- - TABLE Forms of Nitrogen in Various Algae - - C&S Brown Red Green _- - Time of collection Apr. M&l.. Apr. Sept. I _- I Total N in dry plant -_ per cdnt I - 13thanolic extract Total N in I T Total N estimated kid hy- Humin ; lrolysate gcr cent )W GM per cent *cr cmt been due to a decrease in free peptidea during the summer. The humin nitrogen was higher in the brown algae, owing probably to the presence of alginate. The various forms of nitrogen estimated accounted for between 90 and 96 per cent of the total with the exception of Ulva. Some peculiar form of nitrogen must be present in this species, since recoveries of only 80 to 86 per cent have been repeatedly obtained. The distribution of the nitrogen of the amino acids in the five species examined is reported in Table II as percentage of the total nitrogen in the hydrolysate after removal of the humin. The results for Fucus are those previously reported (25) from analyses with both starch and resin columns, with some minor corrections due to revision of color yields. Recovery of nitrogen from the columns ranged from 79 to 89 per cent of the nitrogen in the hydrolysate. These recoveries are comparable to those reported by others for the analysis of plant material containing large amounts of non-protein components. Since both cystine and tryptophan appear to be destroyed during acid

3 D. G. SMITH AND E. G. YOUNG 847 hydrolysis and were not detected on our chromatograms, qualitative tests were applied to the original material. Fresh plants were extracted several times with water at 80 to remove soluble carbohydrate. The final residue was extracted with 5 N sodium hydroxide at 40 for 2 hours. This TABLE Combined Amino Acids in Various Algae The results express nitrogen of the amino acids as percentages of total N in hydrolysate. Amino acid P. ocsicntosus A. nodosum c. crispvst R. )olmato: u. lackca* II Alanine. Arginine... Aspartic acid.. Citrulline.. Cystine. Glycine Glutamic acid.. Histidine Isoleucine Leucine Lysine Methionine.. Ornithine... Phenylalanine.. Proline. Serine. Threonine. Tryptophan. Tyrosine Valine Ammonia. Total * Single determination. t Mean of three determinations; for aspartic acid, threonine, serine, and proline, mean of five. $ Mean of two determinations. extract was adjusted to ph 8 with hydrochloric acid and centrifuged. The supernatant fluid gave a positive qualitative reaction for cystine by the reduced sulfur test and for tryptophan by the Hopkins-Cole test. Both cystine and tryptophan have been reported as present in Rhodymenia and Ascophyllum (4) and in FUCUS, Chow&us, and Ulva (7). Dustin et al. (6) have recently published a report on the determination of amino acids in the presence of large amounts of carbohydrate. In no

4 848 AMINO ACIDS OF MARINE ALGAE instance was the recovery of an amino acid lowered by as much as 3 per cent under the usual conditions of hydrolysis. Only 1 per cent of arginine was lost in this procedure. These authors have observed a red peak with maximal absorption at 520 rnp which emerged well in front of aspartic acid. Reddish peaks with the same point of maximal absorption have also appeared during the analysis of other materials high in carbohydrates (24). A peak with similar characteristics has been observed in the present study. The hydrolysates were colored light brown, and this pigment appeared as a discrete peak near the beginning of the run. In the effluent curves for the basic amino acids in U. Zactuca, there was a small peak immediately preceding histidine. This is considered to be a decomposition product, similar to that noted in the analysis of cassava flour (3), and it has not been included in the calculations. Identification of the amino acids formed on hydrolysis of Chondrus was complicated by the presence of two components which were not found in the hydrolysates of the other algae examined. One unknown appeared in the area between glutamic acid and proline and was largely overlapped by the higher peak due to glutamic acid. The point of separation was indicated by the indentation, which permitted an approximate estimation of this acid. The other unknown preceded and overlapped the lower lysine peak in the effluent curve from the 15 cm. column. From the work of Moore and Stein (21), the first unknown was probably citrulline and the second ornithine. The effluent curves for these particular areas are illustrated in Fig. 1. Subsequently a better resolution of ornithine and lysine was obtained from a 15 cm. column with the buffer sequence recommended by Hamilton and Anderson (14). Elution of the basic amino acids from a 100 cm. column gave rise to a peak in the ornithine position. Chromatography of the hydrolysate was repeated on a 15 cm. column, and the effluent fractions in the lysine area were subjected to a confirmatory analysis for ornithine by the ninhydrin procedure of Chinard (2). The hydrolysates of Rhodymenia and Ulva were also analyzed by this method. The test was positive for ornithine in Chon- &us, but negative in the other two algae. Addition of citrulline to the hydrolysate of Chondrus, prior to chromatographic separation, resulted in a corresponding increase in the second component in the glutamic acid position. The presence of citrulline in the original hydrolysate was confirmed qualitatively by one- and twodimensional chromatography on paper. In order to eliminate the possibility of decomposition of arginine during hydrolysis, a known amount of arginine was added to the residue from an ethanolic extraction prior to hydrolysis. Analysis of the hydrolysate

5 D. G. SMITH AND E. G. YOUNG 849 showed no increase in the content of ornithine. Likewise, hydrolysis of a mixture of arginine and carrageenin did not generate any ornithine or citrulline detectable by chromatographic methods. The possible formation of ornithine and citrulline by enzymic decomposition in the interval between harvesting and drying was investigated. Specimens were picked and frozen immediately in solid carbon dioxide. 2 Chondrus cr~spus 100 cm. Column Dowex 50 $4- s 0 Glu $2- e 3 a s 2 ;0.4- f w $$ Effluent - ml. Chondrus ct~spus I5 cm. Column Dowex 50 e i a Effluent -ml. FIG. 1. Sections of efiluent curves obtained with a hydrolysate of C. crispus on a column of Dowex 50 resin. In the laboratory, the plants were transferred directly from the dry ice into hot 75 per cent ethanol. After several minutes, the specimens were removed and placed in an oven with forced draft at 100 overnight. The material was then pulverized and hydrolyzed with hydrochloric acid, as previously described. It contained ornithine equivalent to 4.7 per cent of the nitrogen of the hydrolysate as well as a small amount of citrulline. The presence of both acids was confirmed by two-dimensional chromatography. The value of 4.7 per cent of ornithine compares favorably with values of 4.7 and 6.3 per cent obtained previously. Isolation of ornithine and citrulline was next attempted. Initial efforts

850 AMINO ACIDS OF MARINE ALGAE at separation of ornithine picrate and of copper citrullinate in crystalline form from the acid hydrolysate were unsuccessful.

6 850 AMINO ACIDS OF MARINE ALGAE at separation of ornithine picrate and of copper citrullinate in crystalline form from the acid hydrolysate were unsuccessful. By the chromatographic procedure of Hirs, Moore, and Stein (15), the basic amino acids in an acid hydrolysate containing 0.6 gm. of nitrogen were resolved on a column of ammonium Dowex 50. The dark soluble humin was eluted at the beginning of the run. The ornithine-lysine fractions were located by tests on aliquots with the Chinard reagent. The combined fractions were freed of buffer by the procedure of Hirs et al. (15), and the residue was dissolved in 10 ml. of 0.5 N hydrochloric acid and diluted to 50 ml. with FIG. 2. X-ray diffraction patterns of (upper) material isolated from a hydrolysate of Chondrus and of (lower) L-ornithine monohydrochloride. a 2 :l mixture of n-propanol and 0.5 N hydrochloric acid. A portion of this solution was passed through a starch column, 7.5 X 20 cm., and the ornithine located as before. The combined fractions were evaporated in vacua almost to dryness. The residue was diluted with 2 N hydrochloric acid and evaporated in vacua to a syrup. The residue was dissolved in warm aqueous ethanol and decolorized with carbon, and the hydrochloride was precipitated with pyridine. It was washed with cold absolute ethanol and recrystallized from aqueous ethanol; melting point (with decomposition) compared to for ornithine monohydrochloride. L-Ornithine hydrochloride was prepared from L-arginine hydrochloride for comparison. X-ray diffraction patterns of this material and that obtained from Chondrus were found to be identical (Fig. 2).

7 D. G. SMITH AND E. G. YOUNG 851 As yet we have not worked out a chromatographic procedure for the separation of citrulline on a preparative scale. DISCUSSION The distribution of amino acids in the proteins of Ascophyllum is similar to that in Fucus. The recovery of nitrogen in the analysis of the Ascophyllum hydrolysate was 10 per cent lower than that for Fucus. This may have been due to a lower recovery throughout the entire analysis, since the majority of the amino acid values are at least 10 per cent lower than the corresponding values for Fucus. It is thus possible that the figures for Ascophyllum should approach more closely the values for Fucus. The presence of purines and pyrimidines from nucleoprotein is a possibility which is being investigated. The proteins of Rhodymenia and Ulwa are also similar in over-all composition, but the combined amino acids in these algae differ in distribution from that of the other three species. The arginine content is slightly higher than that of the brown algae. The basic amino acids predominate over the dicarboxylic acids, which is the reverse of the distribution in the brown algae. The proportion of alanine and glycine is higher, representing 15.4 per cent of the nitrogen in the hydrolysate. The distribution of amino acids in U. lactuca resembles that in C. vulgaris, as determined by Fotiden (11). The major differences are the higher levels of basic amino acids and of proline in Chlorella. It may be of some significance that the levels of the monoaminomonocarboxylic acids in algae and in land plants, such as the spermatophytes, are so similar. Chondrus is remarkable for its high content of arginine and for the presence of citrulline and ornithine in acid hydrolysates of the insoluble material of this alga. The presence of these two amino acids in a combination which is not soluble in 75 per cent ethanol is a distinctive finding. These amino acids have not been recognized heretofore as constituents of proteins. Ornithine occurs in the peptides, tyrocidine (gramicidin S) at 33 per cent (13, 22, ZS), and bacitracin A at 10 per cent of the total nitrogen (5, 17, 18). Any free amino acids or simple peptides should have been removed by the ethanolic extraction in our experiments; therefore the ornithine and citrulline detected must have been in a combined or insoluble state prior to hydrolysis. The presence of ornithine in cassava flour has been explained by the action of bacterial arginase on arginine during retting in manufacture (3). Combined ornithine in gelatin has been proved to be formed from arginine residues by exposure to lime in the course of preparation (14). Citrulline has been estimated chromatographically as constituting 6.3 per cent of the total nitrogen extractable with 75 per cent ethanol from the moss Funaria hygrometrica (19). The occurrence of

8 852 AMINO ACIDS OF MARINE ALGAE arginase in plant tissues appears to be wide-spread, but its function is obscure. Since the amount of ornithine in Chondrus has been found to be relatively constant on repeated analysis, it seems probable that this amino acid must be regarded as a normal constituent of protein or of a complex decomposition product in this alga insoluble in 75 per cent ethanol. The evidence for citrulline is less convincing, in that its presence is based entirely on chromatographic procedures. These have been repeated often enough with positive results to suggest that combined citrulline is also present in Chondrus in small amount. Attempts to fractionate and isolate the protein in Chondrus are in progress at present. The insolubility of the greater portion has made purification difficult, Chromatographic analysis of the ethanolic extracts is also being carried out. SUMMARY The distribution of the combined amino acids in whole plants of five species of marine algae has been determined by quantitative chromatography after extraction with 75 per cent ethanol. The composition of amino acids in the brown algae, Ascophyllum nodosum and Fucus vesiculosus, was very similar. The distribution in the red alga, Rhodymenia palmata, and the green alga, Ulva Zactuca, was also similar, but differed markedly from that in the brown algae. Chondrus crispus was distinctive for its high content of arginine and for the presence of ornithine and citrulline in the acid hydrolysate of the insoluble material of the dried plant. We acknowledge the able technical assistance of Mr. F. G. Mason during the course of this investigation. BIBLIOGRAPHY 1. Channing, D. M., and Young, G. T., J. Chem. Sot., 2481 (1953). 2. Chinard, F. P., J: Biol. Chem., 199,91 (1952). 3. Close, J., Adriaens, E. L., Moore, S., and Bigwood, E. J., Bull. Sot. chim. biol., 36, 985 (1953). 4. Coulson, C. B., Chem. and Ind., 971,997 (1953). 5. Craig, L. C., Hausmann, W., and Weisiger, J. R., J. Biol. Chem., 199, 865 (1952). 6. Dustin, J. P., Ceajkowska, C., Moore, S., and Bigwood, E. J., Anal. chim. acta, 9, 256 (1953). 7. Ericson, L.-E., and Carlson, B., Ark. kemi, 6, 511 (1953). 8. Ericson, L.-E., and SjBstrijm, A. G. M., Acta them. Stand., 6, 805 (1952). 9. Fisher, A. W., Jr., and Burlew, J. S., Carnegie Inst. Washington Pub. No. 600, 303 (1953). 10. Fowden, L., Nature, 167, 1030 (1951). 11. Fowden, L., Biochem. J., 60, 355 (1952).

9 D. G. SMITH AND E. G. YOUNG Fowden, L., Ann. Bot., 18, 257 (1954). 13. Gordon, A. H., Martin, A. J. P., and Synge, R. L. M., Biochem. J., 37,313 (1943). 14. Hamilton, P. B., and Anderson, R. A., J. Biol. Chem., all,95 (1954). 15. Hirs, C. H. W., Moore, S., and Stein, W. H., J. Biol. Chem., 196, 669 (1952). 16. Levy, A. L., and Chung, D., Anal. Chem., 26,396 (1953). 17. Lockhart, I. M., and Abraham, E. P., Biochem. J., 68,633 (1954). 18. Lockhart, I. M., Newton, G. G. F., and Abraham, E. P., Nature, 173,536 (1954). 19. Mansford, K., and Raper, R., Nature, 174, 314 (1954). 20. McFarren, E. F., Anal. C&m., 23, 168 (1951). 21. Moore, S., and Stein, W. H., J. BioZ. Chem., 192, 663 (1951). 22. Sanger, F., Biochem. J., 40, 261 (1946). 23. Schieler, L., McClure, L. E., and Dunn, M. S., Food Res., 18,377 (1953). 24. Schram, E., Dustin, J. P., Moore, S., and Bigwood, E. J., Anal. chim. acta, 9, 149 (1953). 25. Smith, D. G., and Young, E. G., J. BioZ. Chem., 206,849 (1953). 26. Synge, R. L. M., Biochem. J., 39, 363 (1945).

10 THE COMBINED AMINO ACIDS IN SEVERAL SPECIES OF MARINE ALGAE Donald G. Smith and E. Gordon Young J. Biol. Chem. 1955, 217: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1

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