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Max S. Dunn SARDINE, SARDINIA CRULEAACIDS IN THE PROTAMINE OF THEPERCENTAGES OF SOME AMINO

DISTRIBUTION AND THEBASIC PROTEINS: I. THE NITROGENARTICLE:

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BASIC PROTEINS.

I. THE NITROGEN DISTRIBUTION AND THE PERCENTAGES OFSOME AMINO ACIDS IN THE PROTAMINE OF THE

SARDINE, SARDINIA CBRULEA.*

BY MAX S. DUNN.

(From the Chemical Laboratory, University of California, Southern Branch,Los Angeles.)

(Received for publication, August 17, 1926.)

Protamines were discovered in 1874 by Miescher (1) in thespermatazoa of the salmon. Subsequently it has been shownthat protamines are present in the spermatazoa of the carp (2),the common herring (2-8), two species of mackerel (9, lo), threeof sturgeon (3, ll-15), four of salmon (2-5, 11, X-21), and ineight less common species of fishes (22-25). Protamines have

not been found in plants nor in animals other than in the sper-matazoa of fishes.

Our knowledge of the protamines is chiefly due to Kossel andhis coworkers. Their investigations have shown that the pro-tan es are unique as compared to other proteins, particularly inrespect to their amino acid composition. They are the mostbasic of the proteins. In the majority of cases, their basicity isdue to an extremely high content of arginine. With a few ex-

ceptions protamines have been found to contain from about 58to 92 per cent of this amino acid. Lysine, the only amino acidbesides arginine found to an unusual extent in a protamine, con-stitutes 28.8 per cent of a-cyprinine, a protamine of the carp (2).

Protamines are further characterized by the presence of rela-tively few amino acids. In no case has it been proved that aprotamine contains more than five different amino acids (26).

* The writer is indebted to Mr. C. B. Andrews of the California StateBureau of Fisheries, Terminal Island, California, for assistance in the col-lection of the sardine testicles and to Mr. G. L. Samuelson for the prepara-tion of the protamine sulfate used in these experiments.

697

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698 Basic Proteins. I

Because of their high proportion of arginine and low content ofother amino acids Kossel has advanced the assumption that theprotamines are the simplest proteins. Objections to this con-ception have been raised by Abderhalden (18, 27) who believesthat the protamines may be just as complicated as other proteins.

Although the protamines of numerous species of fishes havebeen studied, in only a few cases (28, 29) has quantitative in-

formation concerning their amino acid composition been ob-tained. For this reason and because of the obvious importanceof the protamines to the problems of heredity and to the generalreactions of cells, the present studies were undertaken.

EXPERIMENTAL.

Testicles of the sardine, Sardinia cwulea, were obtained fromthe fish canneries at East San Pedro, California, during the spawn-ing season in May. The ovaries and testicles can readily bedistinguished as the former are yellowish, granular, and coveredwith a network of fine blood vessels while the latter are smooth,grayish, flat, lobular organs adjacent to the kidneys and theventral surface of the fish. The testicles are about 10 cm. inlength in a sardine three times as long.

The testicles were obtained from sardines caught in the Pacificocean about 12 hours previously. They were brought directlyto the laboratory, placed in a cheese-cloth bag, and allowed todrain for several hours in the ice box. The protamine was ex-tracted from the finely ground testicles with 1 per cent sulfuricacid. The protamine sulfate was precipitated with alcohol andpurified according to the procedure of Kossel (3, 11, 23). Thewhite, flocculent precipitate was thoroughly desiccated with al-cohol and ether, and dried in vacua over sulfuric acid. From3015 gm. of the moist testicles 15 gm. of white, powdery pro-tan e sulfate were obtained. This product contained 18.35

per cent of nitrogen (calculated on an ash- and moisture-freebasis).The distribution of nitrogen and the percentages of some amino

acids of the sardine protamine were determined by the VanSlyke method and the calorimetric procedure of Folin and Looney(30). The results of these analyses are given in Tables I, II,III, and IV.

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M. S. Dunn

TABLE I.

Distribu tion of Nitrogen in Four Groups.*

Sardine protamine.Form of nitrogen.

I II Average.

per cent per cent per centHumin N... .... .... .... .... .... ... 0.51 0.52 0.51Amide N.. . . . . . . . . . . . . . . . . . . . . . . . . 0.15 0.16 0.15Basic N........................... 10.26 10.47 10.36Non-basic N.. . . . . . . . . . . . . . . . . . . . . 7.35 7.29 7.32

Total............................ 18.27 18.44 18.34

* Results expressed in percentages of the ash- and moisture-free pro-tamine. Nitrogen content of the protamine, 18.35 per cent.

TABLE II.Distribution ofNitrogen in the Protamine of the Sardine as Determined by the

Van Slyke Melhod .*

Form of nitrogen.

Amide N .......... .......... .Humin N ......... .......... ..Arginine N .... .... .... .... ...Cystine N ......... ......... ..Histidine N .......... ........Lysine N .......... .......... .Amino N of filtrate .... .... ...Non-amino N of filtrate .... ...

Total N regained .... ... ... .

I

07

0.00370.01220.12080.00330.10060.02160.11580.0606

0.4386

-

__

_ -

-

II

07

0.00380.01250.12240.00200.10020.02630.11040.0646

0.4422

-

.-

_1

-

I

per cent

0.852.79

27.650.75

23.034.94

26.5113.87

per cent0.872.86

28.020.46

23.016.02

25.2714.79

.Ol. 30

-

- _

-1

-

Average.

per cent0.862.83

27.830.60

23.02548

25.8914.33

00.84

* Sample I, ash- and moisture-free, 2.39 gm. protamine; 0.4368 gm.nitrogen. Sample II, ash- and moisture-free, 2.39 gm. protamine; 0.4368gm. nitrogen.

A sample of about 6 gm. of the air-dried protamine sulfate was hydro-lyzed ior 26 hours with 250 cc. of 20 per cent hydrochloric acid. Equal ali-quots were taken for analysis.

The total nitrogen regained was corrected for solubility of the bases.The phosphotungstate precipitate was decomposed by the amyl alcohol-ether method.

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700 Basic Proteins. I

TABLE III.

Percentages of Some Amino Aci ds in Sardine Protamine.*

Amino acid. I II Average.

per cent per cent per centArginin e.......................... 15.70 15.91 15.80Cystine ............................ 1.18 0.72 0.95

Histidme.......................... 15.73 15.67 15.70

Lysine............................. 4.71 5.74 5.22

Tyros ine.......................... 1.09 1.09

Tryptophane. . . . . 0.87 0.87

* Percentages based on the ash- and moisture-free protamine. Arginine,

cystine, histidine , and lysine determined by the Van Slyke method.Tyro-

sine and tryptophane determined calorimetrically by the method of Folinand Looney (30). For this purpose a separate 1.0 gm. sample of sardineprotamine sulfa te was hydrolyzed with barium hydroxide for 48 hours.

TABLE IV.

Free Amino Nitrogen of the Sardine Protamine Compared with the LysineNitrogen.*

N gas from5 CG.

One-halfTem pera- Amino N in Ratio of lysine N by

ture. 100 cc.amino N to

total N. Van Slykemethod.

cc. mw 2. C. wn. per cent per cent0.605 750.5 20.5 0.0068 9.98 2.74

* 0.3734 gm. of the ash- and moisture-free protamine was dissolved in

water and diluted to 100 cc. 5 cc. of this solution were u sed for determina-tion of the amino nitrogen in the Van Slyke micro amino nitrogen apparatus.

The total nitrogen in the 100 cc. of solution was 0.0681 gm.

DISCUSSION.

No analysis of the protamine from the sardine has previouslybeen reported. The protamine from only one other member,Cbupea harengus, of the herring (ClupeidE) family has been studied.

The figures for the distribution of amino acids in the prota-mine of the sardine represent maximal values since they wereobtained by the Van Slyke and calorimetric procedures.

The data indicate that the protamine of the sardine, Xardiniaczrulea, differs from that of the common herring, Clulpea harengus.The former protamine is found to contain 15.80 per cent of argi-nine, 15.70 per cent of histidine, 5.22 per cent of lysine, and

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M. S. Dunn 701

smaller percentages of tyrosine, cystine, tryptophane, and am-monia. With the exception of arginine these amino acids arelacking in the herring protamine. Furthermore the latter con-tains 82.2 per cent of arginine. Two other protamines witha low arginine content have been studied. The a-cyprinineof carp contains 4.9 per cent of arginine and the crenilabrinefrom Crenilabrus pavo 23.7 per cent. The protamine from thesturgeon, Accipenser sturio, resembles that of the sardine inhaving histidine and lysine as well as arginine.

Three other protamines besides the sardine have been found tocontain tyrosine (2, 22, 23) and one other tryptophane (26).The low percentage of amide ammonia obtained from the acidhydrolysate of the sardine protamine indicates the presence ofsmall amounts of the dicarboxylic ammo acids, the decomposi-tion of amino acids, or the presence of amino acid anhydrides

(cyclic diacipiperasines) from which ammonia is believed to bederived (31). The presence of cystine in the sardine protamineseems certain since the blue color distinctly obtained with Folinsphenol reagent in the acid hydrolysate of the protamine is specificfor cystine (in the reduced form, cysteine).

An inspection of Table IV shows that the free amino nitrogen,obtained by the reaction of nitrous acid with the native prota-mine, is considerably in excess of one-half of the lysine nitrogen.

These values should be equivalent if, according to a currenttheory, no amino groups other than that on the epsilon carbon oflysine are free in the protamine molecule. A similar disagree-ment has been reported for another protamine. According toKossel and Dakin (2) one-half of the lysine nitrogen from thea-cyprinine of the carp is 15.2 per cent of the total nitrogenwhile Kossel and Cameron (32) found that the free amino nitro-gen of this protamine was 23.6 per cent. Carp protamine wasfound to contain 19.5 per cent of free amino nitrogen by thepresent writer (unpublished results).

It appears, from the results obtained, that six different aminoacids account for about 40 per cent of the amino acids present inthe sardine protamine. Approximately 55 per cent of the totalnitrogen is found in the basic amino acids but the latter consti-tute only about 37 per cent of the protamine. Thus it wouldseem that other monoamino acids, left undetermined by the

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Basic Proteins. I

methods employed, are present. The evidence obtained is notin support of the view that the sardine protamine is of simpleconstruction. Kossels assumption that the protamines are thesimplest proteins may be true in some cases but it appearsun-likely for the sardine, cr-cyprinme of the carp, and for all otherspecies not possessing a high proportion of a single amino acid.

SUMMARY.

1. The protamine sulfate from the ripe testicles of the sardine,Sardinia cmulea, has been purified and obtained as a white powder.Analyzed by the Van Slyke method, the basic ammo acids of theprotamine were found to comprise less than one-half of the totalammo acid content.

2. Data obtained by the Van Slyke method and the Folin andLooney calorimetric procedure indicate that small percentages

of ammonia, tyrosine, cystine, and tryptophane are present inthe sardine protamine.3. The free amino nitrogen obtained from the sardine prota-

mine was in excess of one-half of the lysine nitrogen. This dis-agreement suggests the presence of free amino groups other thanthat on the terminal carbon of Iysine. A similar conclusion isdrawn from the data reported for a protamine of the carp.

4. No evidence is obtained in support of the theory that theprotamines are the simplest proteins.

BIBLIOGRAPHY.

1. Miescher, F., Ber. them. Ges., 1874, vii, 376.2. Kossel, A., and Dakin, H. D., 2. physiol. Chem., 1903-04, xl, 565.3. Kossel, A., 2. physiol. Chem., 1898, 165.xv,4. Goto, M., 2. physiol. Chem., 1902-03, xxxvii, 94.5. Kossel, A., and Dakin, H. D., 2. physiol. Chem., 1904,xli, 407.6. Mathews, A., 2. physiol. Chem., 1897, xxiii, 399.7. Kossel, A., 2. physiol. Chem., 1898-99, xxvi, 588.8. Gross, E., 2. physiol. Chem., 1922, 167.xx,9. Kurajeff, D., 2. physiol. Chem., 1898-99, xxvi, 524.

10. Sumikura, K., J. Tokyo Chem. Sot., 1920, xli, 281; abstracted in Chem.Abet., 1920, xiv, 2805.

11. Kossel, A., 2. phys iol. Chem ., 1896-97, xxii, 176.12. Kossel, A., andDakin, H. D., 2 physiol. Chem., 1905, xliv, 342.13. Malentik, W. D., 2. physiol. Chem., 1908, Ivii, 99.14. Kossel, A., and Weiss, F., 2. physiol. Chem., 1912, lxxviii, 402.

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Id. S. Dunn 70315. Kuraje ff, D., Z. ph~ siol. Chem ., 1901, xxxii, 197.16. Piccard, J., Ber. them. Ges., 1874, vii, 1714.17. Ko sscl , A., 2. physiol. Chem., 19OS-04, xl, 311.

18. Abderhalden, E., Z. phgs iol. Chem., 1904, xli, 55.19. We iss, F., Z. physiol. Chem., 1907, lii, 107.20. Taylor, A. E., J. Biol. Chem., 1906-09, 389.,

21. Lynch, V., J. Biol. Chem., 1920, xliv, 319.

22. MorkoFin, h., 2. ph?/sioZ. C&m ., 1899, xxviii, 313.

23. Ko sse l, A., Z. p71~~siaZ. Chem., 1910, Ixix, 138.24. Kos sel, A., 2. physiol. Chem., 1913, lxxxviii, 163.25. Ko sse l, A., and Edlbacher, F., Z. phys iol. Ckem., 1913, lxxxviii, 186.26. Ko sse l, ,4., Z. ph?ysioZ. Chcm ., 1905. xliv, 347.

27. Abderhalden, E., A textbook of physio logical chemistry, translated byHa ll, W . T., and Defren, G., New York, 1914, 136.

28. Plimmer, R. H. A., The chem ical constitution of the proteins, Part I,

Ana lysis, New York, 3rd edition, 1917, 111.29. Mathews, A. P., Phys iological chemistry, New York, 2nd edition, 1916,

128.

30. Folin, O., andLooney. J. i\I., J. Biol. Chem.. 1922, Ii, 421.31. Dunn, M. S., J. Am. Chcm . S ot., 1925, xlvii, 2564.32. Ko sse l, A., and Cameron, A. T., Z. physiol. Chem., 1911-12, lxxvi, 457.