THE OCCURRENCE OF AMINO ACIDS AND OTHER OR- GANIC NITROGEN COMPOUNDS IN LAKE WATER.*

Transcription

1 THE OCCURRENCE OF AMINO ACIDS AND OTHER OR- GANIC NITROGEN COMPOUNDS IN LAKE WATER.* BY W. H. PETERSON, E. B. FRED, AND B. P. DOMOGALLA. (From the Wisconsin Geological and Natural History Survey and the University of Wisconsin, Madison.) (Received for publication, January 26, 1925.) Although little is known regarding the nature of the soluble organic nitrogen in surface waters, there is no doubt but that it contains compounds of great biological significance to microorganisms, plants, and animals. The superiority of organic forms of nitrogen as compared with inorganic compounds for the nutrition of the majority of bacteria and related microorganisms is well known. Likewise in the growth of plants, water cultures containing amino acids, according to Schreiner and Skinner (l), gave better results than nutrient solutions containing only inorganic compounds of nitrogen. For a number of years, the reports of Putter (2) have been the center of a controversy as to whether or not aquatic animals can utilize the organic nitrogenous compounds dissolved in the water. A knowledge of the nature of the organic nitrogenous compounds contained in surface waters would be of value in determining whether Putter s theory is within the realm of possibility. Obviously, if the water does not contain such indispensable amino acids as tryptophane, cystine, tyrosine, and histidine, it cannot furnish the nutrients required by aquatic animals. In the preceding paper (3) it was shown that lake and river waters contain considerable quantities of organic nitrogen which can be precipitated by phosphotungstic acid and other protein precipitants. It was also found that this organic nitrogen contained amino, peptide, * This work was supported jointly by grants from the United States Bureau of Fisheries and the special research fund of the University of Wisconsin. 287

2 288 Nitrogen Compounds in Lake Water and non-amino nitrogen. In this paper, data will be presented to establish the existence of amino acids, purines, and other forms of organic nitrogen in such waters. EXPERIMENTAL. Lakes Studied.-Four inland lakes of Wisconsin and Lake Michigan have been included in this study. A detailed description of the Wisconsin lakes and their plant and animal life is given in Bulletins 27 and 64 of the Wisconsin Geological and Natural History Survey. Only a brief statement will be given here regarding each lake. Lake Mendota, located at Madison, is 6 miles long, 4 miles wide, and has a maximum depth of 87 feet. It is a hard water lake and contains an abundant growth of plankton. Green Lake is also a hard water lake, about the same size as Mendota, but has a much greater depth, 225 feet. The plankton growth is somewhat different and also less abundant than in Mendota. Devil s Lake is a soft water lake, smaller and shallower (depth 43 feet) than Mendota and, compared with the latter, is relatively poor in plankton. Turtle Lake is located in the cut-over timber region of northern Wisconsin and has highly colored water. It is a soft water lake about the size of Devil s Lake and contains essentially the same type of plankton. Lake Michigan contains only a small growth of plankton as compared with the Wisconsin lakes. The sample was taken at the North Side Pumping Station intake for the city of Milwaukee. This intake is about 1 mile from shore, and 65 feet below the surface. The depth of the lake at this point is about 300 feet. Preparation of the Samples.-Surface sa.mples were taken from all of the Wisconsin lakes and a bottom sample from Mendota. The bottom sample was taken 65 feet below the surface where the lake is about 75 feet deep. From 200 to 600 liters of water were brought to the laboratory for analysis. The samples other than those from Mendota were taken in the late fall or winter and were kept cold en route. In most cases less than 10 hours and in no case more than 24 hours elapsed between the time of sampling and the beginning of the analysis. On arriving at the laboratory the sample was immediately run through a high speed Sharples centrifuge at 40,000 R.P.M. which removed the plankton, including 70 to 80 per cent of the bacteria. The clarified water was concentrated in a vacuum pan at 60 C. to a small volume, 200 to 500 cc., and finally evaporated to dryness with a fan at 65 C. Analyses of the concentrated water from

3 Peterson, Fred, and Domogalla 289 the vacuum pan and the dried residues showed the same percentage of amino and other forms of nitrogen. It is felt, therefore, that there was no appreciable change in the forms of nitrogen as a result of evaporation of the concentrates to dryness. Qualitative Test for Proteins and Amino Acids.-Two Mendota samples and one each from Devil s Lake and Green Lake were tested qualitatively for proteins and amino acids by various reagents (4-9). When applied directly to the water concentrate, the reagents did not in general give good tests. Hydrolysis with hydrochloric acid or barium hydroxide hydrolysis and separation of the inorganic salts gave better results. The ninhydrin, Folin and Looney, and Inouye reagents gave strong TABLE I. Protein Tests Given by Samples of Concentrated Lake Waters. Treatment and reagent. Gies biuret Fair. Millon s, tyrosine Xanthoproteic , Bromine, tryptophane Hopkins-Cole s, tryptophane.... Danila s, tryptophane , May and Rose s, tryptophane Inouye s, histidine ,. Good. Harding and Warneford s, ninhydrin. Folin s, amino nitrogen Good. Fair. Good. Fair. Good. Fair. Fair. Good. Fair. G color tests on the concentrated water as well as on the hydrolyzed solutions. The results are given in Table I and show that all of the samples gave positive tests with most of the reagents. Positive results with such a number of tests point strongly to the presence of proteins or amino acids in these surface waters. Quantitative Determination of Certain Amino Acids. Tryptophane.-This amino acid was determined by the methods of Ftirth and Nobel (lo), Folin and Looney (ll), and May and Rose (7). The first method was modified somewhat by precipitating the tryptophane with mercuric sulfate as in the Folin and Looney procedure. Hydrochloric acid was used to dissolve

4 290 Nitrogen Compounds in Lake Water the precipitate instead of sodium cyanide, as the latter chemical destroys the color produced by tryptophane with the Fiirth and Nobel reagents. Dried lake samples equivalent to 50 liters of water were hydrolyzed with Ba(OH)2 (14 gm. Ba(OH)2*8Hz0 per 100 cc.) for 20 hours,in an autoclave at 15 pounds pressure (121 C.). Longer heating (30 hours) did not increase the quantity of amino nitrogen. Upon cooling, a large proportion of the inorganic salts settled out. The insoluble material was filtered off, washed with saturated Ba(OH)2, and the barium removed with 28 per cent H2SOh. The filtrate and washings from the BaS04 were concentrated to 50 cc. and the tryptophane determined in 6 cc. aliquots, following the detailed directions of the authors. As a check on the procedure a known quantity of tryptophane was added to another aliquot of the water sample and the total tryptophane content determined in the usual manner. As complete recovery of the added amino acid was obtained, it is felt that the results are a correct measure of the tryptophane content of the water. The figures obtained by three independent methods check within reasonably close limits. Tyrosine.-Another aliquot of the hydrolysis products was used for the determination of tyrosine according to the Folin and Looney procedure. That reliable results were secured was attested by the complete recovery of known quantities of tyrosine when added to the unknown solution. Cystine.-Since cystine is destroyed by heating with alkali, the dried lake water residues equivalent to 20 liters were boiled with 20 per cent HCl for 12 hours instead of Ba(OH)2. The peptides were completely hydrolyzed at the end of that time as judged by amino nitrogen determinations made during the hydrolysis. The acid was removed in a vacuum and the concentration continued until the volume was less than 50 cc., when the solution was transferred to a 50 cc. flask and made up to volume. Aliquots were analyzed for cystine by the Folin and Looney method. A strong blue color developed in each case and no difficulty was experienced in matching it with the standard. When known quantities of cystine were added to the hydrolyzed samples, the increase in cystine found was exactly equivalent to that added.

5 Peterson, Fred, and Domogalla 291 H&%&n~.-Histidine was determined in the acid-hydrolyzed samples by the Koessler and Hanke (12) method as adapted by the authors to urine. Some further modifications were necessary in order to make it applicable to lake water residues. Since tyrosine was present and was found to give a color reaction with the diazobenzenesulfonic acid reagent, it had to be removed completely. Preliminary experiments with solutions containing tyrosine and histidine showed that tyrosine can be separated quantitatively from the histidine by four precipitations with Koessler and Hanke s lead acetate and sodium hydroxide reagents. Rapid sedimentation of the precipitate was obtained Amino acid. Method. Tryptophane. Tyrosine. Histidine. Cystine. TABLE Quantity of Certain Amino Acids Found in Some Lake Waters. 3x z %d 3s 29 VI - il - cafz?s g 2s 9: -2F s ci - ml. m7. mg. w?. m7. xi.. n cu. m P@- m. n? :u m Ftirth and Nobel ,: 12.: Folin and Looney. 11.c 14.f 12.g May and Rose. 9.c 12.: Folin and Looney ! 17.c Koessler and Hanke : Folin and Looney. 1.E 6.: 3.: Total organic nitrogen II. m?. cl& m by the use of a centrifuge. After the lead was removed with disodium hydrogen phosphate the filtrates were concentrated to 2 cc. and the histidine was determined according to the first method given by Koessler and Hanke. The calorimeter readings were exactly the same as those given by untreated solutions of histidine dichloride. Complete recovery was also obtained of known quantities of histidine added to lake samples. The quantitative data obtained by the methods just described are given in Table II and show that all of the lakes contained appreciable quantities of the four amino acids. The average for tryptophane, tyrosine, and histidine is about 13 mg. and for cystine about 4 mg. per cubic meter of water.

6 292 Nitrogen Compounds in Lake Water The surface water of Mendota contains less amino acids than does the bottom sample. Certain amino acids are high in some lakes and low in others. No direct comparisons can be made as the samples were not taken at the same time of year nor in the same year. A long series of analyses of Mendota water has shown that there is great variation in the nitrogen content from season to season and from year to year. The type and quantity of plankton growing in the lake are important factors in determining the kind and amount of amino acids in solution at any given time. Since the soluble nitrogen is derived in part from the plankton it is to be expected that the forms of nitrogen will vary. Other Forms of Nitrogen. As plenty of material was conveniently available, some other forms of nitrogen were determined on the surface and bottom samples of Lake Mendota. Free Amino Nitrogen.-The free amino nitrcigen was determined on 10 cc. aliquots of the concentrated water by means of the Van Slyke (13) apparatus. For this purpose the regular apparatus was replaced by a special burette which combined the large bulb of the macro apparatus and the capillary tube of the micro apparatus. Amides.-Aliquots of the concentrated water equivalent to 200 liters of the original water were hydrolyzed for 12 hours with 20 per cent HCl. After hydrolysis the acid was removed in vacuum as directed by Van Slyke. The residue was made alkaline with a 10 per cent suspension of lime and the amide nitrogen distilled off in vacuum at 65 C. into standard acid. The residue in the distilling flask was filtered, washed until free from chlorides, and the humin nitrogen determined in the insoluble material by the Kjeldahl method. Peptide Nitrogen.-The filtrate and washings were concentrated to a small volume and amino nitrogen was determined in an aliquot by the Van Slyke method. The difference between the amino nitrogen before and after hydrolysis is called peptide nitrogen. Purines.-The remainder of the solution, after the determination of peptide nitrogen, was used for the determination of total

7 -- - Peterson, Fred, and Domogalla 293 purines by Benedict and Saiki s (14) modification of Kreuger s method. Arginine.-After the precipitation of the purines, the filtrate and washings were concentrated in a vacuum to 200 cc. and boiled 6 hours with 40 gm. of KOH in an apparatus similar to that described by Koehler (15). The arginine nitrogen was calculated from the resulting liberated ammonia. III NO. II I -_ Plankton... TABLE III. Forms of Nitrogen in Mendota Water. Soluble nitrogen Free ammonia nitrogen.. 2. Residual ammonia nitrogen.. 3. Nitrite nitrogen Nitrate.. 5. Free amino nitrogen Peptide nitrogen..... :. 7. l/2 of tryptophane nitrogen /3 of histidine. 9. 3/4 of arginine. 10. Amide nitrogen Purine Amine..:. Forms of soluble nitrogen determined, total.... Surface June 18, w?. cu. m. ;: cu. 7% Bottom June 25, mg. cu. m IV Undetermined nitrogen / Amines.-Nitrogen in the form of amines was determined by the method of Weber and Wilson (16) in aliquots equivalent to 10 liters of lake water. Since the quantity of ammonia was small it was Nesslerized instead of titrated. Ammonia, Nitrites, and Nitrates.-These forms of nitrogen were determined directly on the lake water according to the Standard Methods of Water Analysis (17). A small quantity of

8 294 Nitrogen Compounds in Lake Water ammonia remains in the concentrated water sample which can be obtained by Folin s aspiration method. This is called residual ammonia, while that obtained by simple distillation is called free ammonia. The complete analyses of the surface and bottom samples of Mendota water are given in Table III. Of the total nitrogen, approximately 90 per cent is accounted for by the different forms determined. The bottom water runs uniformly higher in all forms of nitrogen than the surface sample. It is conspicuously higher in a,mmonia, nitrate, and amino nitrogen. The production of soluble nitrogen takes place principally at the bottom of the lake and is brought about by the action of bacteria on the plant and animal debris which accumulates there. At the time of year when these samples were taken, the lake is stratified into three zones with practically no mixing between the top and bottom layers. In the bottom zone a large production of ammonia and nitrates by nitrifying bacteria and but little consumption of these compounds by plant forms take place. In the surface water the conditions are reversed. The plant and animal life in the bottom stratum is less abundant than in the surface zone and hence the plankton nitrogen is much lower. The humin nitrogen in the surface and bottom samples a- mounted to 10.8 and 14.3 mg., respectively. It is approximately the same as that for tryptophane and is in line with the results of Gortner and his associates on the production of humin nitrogen from tryptophane during the hydrolysis of proteins. It is further evidence of the existence of tryptophane in lake waters. SLJMMARY. The presence of proteins and amino acids in lake waters has been established both by qualitative and quantitative methods. Large samples of water from different types of Wisconsin lakes and also from Lake Michigan were concentrated and tested for proteins by ten different reagents. In all cases positive results were secured. The quantity of tryptophane, tyrosine, histidine, arginine, and cystine was determined in each water. The average of the first three amino acids was about 13 mg. and for cystine about 4 mg. per cubic meter of water.

9 Peterson, Fred, and Domogalla The quantity of amine, amide, and purine nitrogen was determined in top and bottom water samples from lake Mendota. These forms of organic nitrogen were found to be more abundant in the lower than in the upper stratum. The soluble nitrogen was separated into twelve different forms and the quantity of each determined. In this way about 90 per cent of the total can be assigned to different fractions. BIBLIOGRAPHY. 1. Schreiner, O., and Skinner, J. J., U. S. Dept. Agric., Bureau of Soils, Bull. 87, Putter, A., Die Ernahrung der Wassertiere, Jena, 1909; Biol. Cents., 1922, xlii, Domogalla, B. P., Juday, C., and Peterson, W. H., J. Biol. Chem., 1925, lxiii, Hawk, P. B., Practical physiological chemistry, Philadelphia, 7th edition, 1921, , Danila, P., Compt. rend. Sot. biol., 1923, Ixxxviii, Inouye, K., 2. physiol. Chem., 1913, lxxxiii, May, C. E., and Rose, E. R., J. Biol. Chem., 1922, liv, Harding, V. J., and Warneford, F. H. S., J. Biol. Chem., 1916, xxv, Folin, O., J. Biol. Chem., 1922, Ii, Ftirth, O., and Nobel, E., Biochem. Z., 1920, cix, Folin, O., and Looney, J. M., J. Biol. Chem., 1922, li, Koessler, K. K., and Hanke, M. T., J. Biol. Chem., 1919, xxxix, 497. Hanke, M. T., and Koessler, K. K., J. Biol. Chem., 1924, lix, Van Slyke, D. D., J. Biol. Chem., x, Benedict, 8. R., and Saiki, T., J. Z3ioZ. Chem., , vii, Koehler, A. E., J. BioZ. Chem., 1920, xlii, Weber, F. C., and Wilson, J. B., J. BioZ. Chem., 1918, xxxv, Standard methods for the examination of water and sewage, Am. Pub. Health Assn., New York, 5th edition, 1923,

10 THE OCCURRENCE OF AMINO ACIDS AND OTHER ORGANIC NITROGEN COMPOUNDS IN LAKE WATER W. H. Peterson, E. B. Fred and B. P. Domogalla J. Biol. Chem. 1925, 63: 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