THE DETERMINATION OF FREE AMINO NITROGEN IN PROTEINS.

BY D. WRIGHT WILSON.

(From the Laboratory of Physiological Chemistry, Johns Hopkins Medical School, Baltimore.)

(Received for publication, March 2, 1923.)

In an effort to determine the free amino nitrogen in Bence- Jones protein by the method of Van Slyke (l), it was observed that the protein was precipitated as soon as it entered the reacting chamber. As Bence-Jones protein is relatively insoluble in weakly acid solution, the possibility of an incomplete reaction between the free amino groups and the nitrous acid made it advisable to check the analysis by the form01 titration method of SGrensen (2). This led to a comparison of the two methods.

Both methods have been used extensively in the past for the determination of free amino groups. While each has seemed to be fairly satisfactory, difficulties arise in their application under certain conditions. When the individual amino-acids are studied, it is found that, with few exceptions, the same grouping is deter- mined by both methods; i.e., the or-amino nitrogen. The E- amino nitrogen of lysine is also determined although it reacts somewhat more slowly in Van Slyke’s procedure than do a- amino groups. The nitrogen of proline is determined by Siiren- sen’s method, but not by Van Slyke’s. Some anomalous reac- tions are obtained. Glycocoll and cystine give more than the theoretical quantity of gas by Van Slyke’s method. SCirensen obtained values higher than the theoretical with tyrosine and lower with histidine, proline, and lysine.

In Sijrensen’s method the reaction of the protein solution is first adjusted so that the free amino groups of the protein are just neutralized by acid groups, then formaldehyde is added and, by the formation of methylene-amino derivatives, the basic property of the amino groups is destroyed and there is liberated

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192 Amino Nitrogen in Proteins

thereby an equivalent amount of acid which is subsequently titrated. The method has the inherent disadvantage of de- pending upon the titration, between rather arbitrarily chosen end-points, of mixtures of weakly acidic and basic groups of unknown strength and variable concentration. Theoretically the titration should be made from the isoelectric point of the protein to the end of the titration curve of the compound formed by formaldehyde. But even without the exact conditions of titration defined, the method nevertheless yields results which are probably not far from the theoretical.

In Van Slyke’s method the uncertainties of the above titration are overcome by actually measuring the gas liberated by the interaction of the amino groups with nitrous acid, but other difficulties are encountered. Most proteins or their deaminized products precipitate in the nitrous acid solution. It would seem, therefore, that occlusion of some of the material not yet acted upon must occur and cause an increase in the length of time necessary for complete reaction. Van Slyke and Birchard (3) recognized this difficulty and attempted to overcome it by increasing the time of reaction from 2 to 5 minutes to 20 to 30 minutes. Owing to the lack of theoretical values to show when the reaction is complete and owing to the possibility of some hydrolysis of the protein occurring during the time of reaction, it is uncertain whether or not this procedure yields correct results.

Several investigators, working mainly with digestion products of proteins, have compared the two methods. White and Thomas (4) found that the results with Van Slyke’s method (apparently using the 5 minute period of reaction) were parallel with, but lower than, those with Sorensen’s method. Rogozinski (5) and Andersen (6) noticed variations with the Van Slyke method and concluded that Sorensen’s method was more satisfactory. Kossel and Cameron (7) obtained different quantities of nitrogen with Van Slyke’s method when sturine was allowed to react for different lengths of time, but later Kossel and Gawrilow (8) made use of the form01 titration.

Abderhalden and Kramm (9), in analyzing digestion mixtures of proteins by Van Slyke’s method, found that great differences in results were obtained depending on whether the reaction was allowed to continue 5 minutes or 10 and suggested that some

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D. Wright Wilson 193

easily split peptones might be hydrolyzed. They were cognizant of the fact that no splitting had been noticed in previous work on pure polypeptides, but considered that these data were in- sufficient. By running comparative experiments with the form01 titration method they found that Van Slyke’s method yielded lower results after a reaction of 5 minutes and higher results after a reaction of 10 minutes and suggested that Sorensen’s method should be used to standardize the time of reaction of the Van Slyke procedure.

Northrop (10) states: “For absolute determinations of the amino-acids Van Slyke’s method is more accurate, for compara- tive experiments concerning the changes occurring in gelatin solutions, such as were used in this work, the form01 titration is more accurate and also much more rapid.”

The possibility of hydrolysis of the protein during the deter- mination was considered by Van Slyke and Birchard (3) who concluded that none occurs because:

“1. Peptides of varied composition and containing up to fourteen amino-

acids in the molecule have been analyzed by our method and found to give theoretical results.

“2. The evolution of nitrogen is complete inside of twenty or thirty

minutes, following practically the course found in analysis of lysine. . . . .”

Data concerning the latter statement are considered below. Hydrolysis can only be studied satisfactorily by eliminating a

confusing element in the determination of native proteins; namely, precipitation by nitrous acid in the reacting chamber. Peptone and proteose solutions are useful in this connection as they yield little or no precipitate under the conditions of the experiment. Rice (11) reported a few experiments with peptone to show that not only is more nitrogen obtained in 8 than in 6 minutes, but that the temperature at which the reaction is carried out in- fluences the results, more nitrogen being obtained at the higher temperature. The difference observed between the highest and lowest results is too great to be accounted for by the delayed reaction of the e-amino group of lysine, and suggests that there may have been a slight progressive hydrolysis.

Hydrolysis may not be the only factor involved in the increase of nitrogen with the lengthening of time of reaction. Slowly

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194 Amino Nitrogen in Proteins

reacting groups may possibly contribute to the high results. Kossel and Cameron reported that arginine and nitroarginine give off increasing quantities of nitrogen as the time of reaction is increased, quantities which are above the theoretical for one reacting nitrogen. They also found that nitroguanidine gives off small but increasing quantities of nitrogen in the reactions. The author has consistently obtained quantities of gas equivalent to 1 per cent of the total nitrogen by allowing guanidine and methyl guanidine to react for 4 to 1 hour.

It would appear from this literature that Van Slyke’s procedure may possibly yield high results due to hydrolysis and other factors, or low results due to incomplete reaction of the precipi- tated protein. Sorensen’s method on the other hand is not free from theoretical objections. The method of Harding and Mac- Lean (12) seems to be liable to greater errors.1 Parallel deter- minations were therefore made on various solutions of proteins using the methods of Van Slyke and of Sorensen for comparison.

The micro apparatus of Van Slyke was used in these analyses. The periods of time allowed for the reaction were based on the suggestion of Van Slyke (14) ; namely, 3 minutes for temperatures between 20 and 25’; 2$ minutes between 25 and 30”; and 2 minutes above 30”. Besides these minimum periods of reaction, samples of each solution were allowed to react twice and five times as long. When determinations involving the longest period were carried out, the reacting chamber was shaken vigorously at intervals of about 1 minute to keep the contents well mixed. The blanks for the various reagents varied in different experi- ments either due to the use of different quantities or different preparations of octyl alcohol. The blanks are not specifically reported, but may be found by subtracting the corrected from the observed readings given in the tables.

The method of Sorensen was carried out as follows: A portion of the protein solution was titrated to pH 7.0 with neutral red as an indicator, using a standard solution for comparison. To another portion of 20 cc. were added 10 cc. of neutralized formalin (40 per cent) and 6 drops of 1 per cent phenolphthalein and the solution was titrated with 0.1 N sodium hydroxide to a deep red

1 Folin’s method (13) was not available when these experiments were

carried out.

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D. Wright Wilson 195

color. The end-point was matched against the color produced by mixing 20 cc. of neutral boiled water, 10 cc. of neutralized formalin, 0.3 cc. of 0.1 N sodium hydroxide, and 6 drops of 1 per cent phenolphthalein. Where the original protein solution was colored, the standard was placed behind some of the protein solution, similarly diluted, in a comparator and the resulting color was used as the end-point for the titration. From these titrations the quantity of alkali necessary to titrate the solution from pH 7.0 to the end-point chosen was determined, and, after subtracting the 0.3 cc. blank, the amino nitrogen per cubic centimeter was calculated. The total nitrogen was determined by the Kjeldahl method. Duplicates were run in all analyses unless otherwise stated.

Various preparations of protein material were studied, but no attempt was made to get preparations of highest purity with which to make the comparisons. The following materials were used :

1. Commercial Peptone. 2. Proteose 1 .-This was prepared from beef which had been digested with

pepsin, by precipitating with ammonium sulfate and removing the salt by boiling with barium carbonate. The filtered solution was concentrated, precipitated with alcohol, and dried with alcohol and ether.

Proteose 8.-A portion of Preparation 1 was reprecipitated twice with ammonium sulfate and evaporated in VCKUO with barium carbonate several times. When free from ammonia it was precipitated with alcohol and dried

with alcohol and ether. These preparations gave no test for ammonia by shaking with permutit, and treating it with sodium hydroxide and Nessler’s solution.

3. Egg Albumin, Crystalline.-The egg albumin crystallized three times,

dialyzed, and dried. A trace of ammonia was removed by shaking with permutit .

4. Egg Albumin, Puri$ed.-Egg white was diluted with 5 volumes of

water and filtered. The albumin was twice precipitated by saturating the solution with sodium sulfate. The final precipitate was dissolved in water and analyzed.

6. Serum Globulin.-This was roughly prepared from pig serum by

precipitation with sodium sulfate. The globulin precipitate was washed and reprecipitated. The moist precipitate was dissolved and analyzed.

6. Edestin.-Edestin was prepared from hemp seed and crystallized three times. It was dried with alcohol and ether, and dissolved in 10 per cent sodium chloride.

7. BenceJones Protein (No. R 5).-This was twice precipitated with

sodium sulfate and acetic acid and dried with alcohol and ether. This

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Amino Nitrogen in Prot,eins

preparation (No. It 5) was isolated from urine which had been preserved

with toluene and allowed to stand for 2 or 3 months at room temperature.

In Table I will be found data obtained by analyzing the various solutions of protein material with the methods of Sorensen and of Van Slyke. With the latter method the reactions were permitted to continue for various lengths of time. It is at once apparent that there is considerable disagreement between the two groups of data. There is a continuous increase in the values obtained by Van Slyke’s method as the period of reaction is lengthened and no single period of reaction yields results with all of the preparations similar to those obtained by Sorensen’s method.

TABLE I.

Amino nitrogen.

,

SiiMP Van Slyke method.

-__-~- m*. w. WT. ml7. WT.

pa-cc. percc. percc. perec. perce.

Peptone ._._. _. . 0.730 0.648 0.712 0.762 0.790 “ * . . . . . . . . . . . . . . . . . . . . . . . . . . 0.180 0.167 0.188

Proteose 1. _, ,__. _. . . . 0.170 0.158 0.182 0.191 0.200

Egg albumin (crystalline). . . . . 0.127 0.063 0.091 0.104 “ “ . . ; .

Serum globuhn’.‘. 1: : : : : . . 0.155 0.077 0.107 0.145 0.189 0.137 0.170 0.205

Edestin................................ 0.052 0.042 0.045 0.059 Bence-Jones Protein5.................. 0.138 0.069 0.097

* The first solution diluted with 3 volumes of water.

Periods of reaction of 8 to 10 minutes and 20 to 30 minutes have been suggested for analyzing protein material by Van Slyke’s method and, as mentioned above, Abderhalden and Kramm used the method of Sorensen to standardize the period of reaction. In the present experiments with the native proteins, edestin and serum globulin, the 4 to 6 minute period of Van Slyke’s method yielded lower results than Sorensen’s method. With the egg albumin preparations, however, even the 10 to 15 minute periods of Van Slyke’s method yielded results lower than the Sorensen procedure. The Bence-Jones protein reacted at a similar rate. On the other hand, the proteose and the dilute

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D. Wright Wilson

peptone solutions gave higher results in 4 to 6 minutes. These data do not support the idea that any single period of reaction in Van Slyke’s method yields results similar to Sorensen’s for all types of protein material.

As Van Slyke has observed, the native proteins were precipi- tated as soon as their solutions were mixed with the acid nitrite solution. This precipitation probably decreased the rate of reaction. In order to det’ermine whether or not the continuous rise in values as the time of reaction increased was due merely to the delayed reaction of material occluded in the precipitate, the solutions of peptone and proteose were carefully studied. The peptone did not precipitate in the reacting chamber and the proteose formed first a turbidity and then, as the time of reaction continued, a slight precipitate. It will be seen that in spite of the lack of a precipitate to retard the action, the values for amino nitrogen increased continuously as the time of reaction was lengthened.

It should be noted, however, in studying the rising values that the c-amino group of lysine reacts more slowly than the LY- amino groups of the amino-acids. Some of the free amino nitrogen in the proteose and peptone solutions is undoubtedly c-amino nitrogen of lysine. Van Slyke first demonstrated quantitatively that c-amino groups of lysine alone are uncombined in native proteins, while in proteoses and peptones, other amino groups are free. As the data reported in this paper show that th:: pep- tone preparation contained about 27 per cent of the total nitrogen in the form of free amino nitrogen, the proteose about 8 per cent, and the native proteins 2 to 5 per cent, one may assume that about half of the free amino nitrogen of the proteose and about one-sixth of the free amino nitrogen of the peptonc is E-amino nitrogen of lysine.

Considerable data concerning the rate of reaction of the E- amino group of lysinc are now available. Van Slyke reported that at 24”, 95 per cent of the nitrogen reacts in 5 minutes and 100 per cent reacts in 15 minutes; at 20” it reacts completely in 30 minutes. Sure and Hart (15) showed that the reaction is greatly influenced by temperature and that it is complete in 5 minutes at 32”. Dunn and Schmidt (16) have recently re- ported that the epsilon group of lysine reacts completely in 8 minutes at temperatures between 26 and 30”.

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198 Amino Nitrogen in Proteins

Solutions of peptone were studied by Van Slyke’s method at temperatures above 30” and in periods of 2, 4, and 20 minutes. A single determination was made using a reaction time of 30 minutes. At the beginning of Table II may be found the individ- ual analyses of these solutions. The reaction should have been nearly complete in 2 minutes at 31” as most of the reacting nitro- gen was a-amino nitrogen. It will be observed that the values rise steadily. A small quantity of ammonia was present in this preparation, but it is doubtful if the continuous increase can be ascribed to it. Values for the 10 and 30 minute periods are higher than those of Sorensen’s method with which ammonia is determined completely. A much more probable explanation is that a slight hydrolysis occurs in the Van Slyke procedure yielding results which are too high.2 This peptone solution was diluted with 3 volumes of water and the diluted solution reacted more rapidly than the stronger solution, yielding results in 4 minutes practically as great proportionately as yielded by the stronger solution in 10 minutes. Sorensen’s method yielded the expected values with the diluted solution, a value slightly less than that given by Van Slyke’s method in 4 minutes.

Proteose solutions yielded results similar to the peptone solu- tions. The values increased as the time of reaction was length- ened. Solution 1 in 6 minutes gave higher results by Van Slyke’s. method than by Sbrensen’s. Solution 2, which was more dilute and was analyzed at a higher temperature yielded values which increased even more rapidly. If the 30 minute period is assumed to yield the correct result, then in 2 minutes only 60 per cent of the amino nitrogen had reacted instead of 90 to 95 per cent which would be expected from reactions of amino-acids and polypep- tides. Such a calculation is obviously incorrect and leads to the conclusion that the long periods of reaction yield results which are too high. A slow continuous hydrolysis appears to be the cause of the high values.

2 Another explanation, of course, is that there are unknown groups in these preparations which react slowly with nitrous acid at a rate even slower than that of the E-amino group of lysine. These slowly reacting groups are presumably not determined by Sorensen’s method because the

results obtained with this method are lower than the maximum values observed with Van Slyke’s method.

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D. Wright Wilson 199

TABLE II

Time. Temperature. Reading. I Corm-ted reading.

Peptone.

2 cc. of 2 per cent solution used with 0.10 cc. ocbyl alcohol. Barometer 755 mm. Hg.

mzn. “cf. cc. cc. mg. pm cent

2 30 2.54 2.42 0.648 82

4 31 2.80 2.67 0.712 90

10 31 3.00 2.86 0.762 96

30 30 3.13* 2.95 0.790 100

2 cc. of 0.5 per cent solution used with 0.10 cc. octyl alcohol.

Barometer 754 mm. Hg.

2 4

Proteose. 1.5 per cent Proteose 1, 2 cc. used with 0.10 cc. octyl alcohol.

Barometer 760 to 762 mm. Hg.

3 25.5 0.69 0.57 0.155 1 79

6 26.5 0.79 0.66 0.182 91

15 j

26.0 0.83 0.69 0.191 95

30 27.5 0.91* 0.73 0.200 100

Proteose 2, 2 cc. used with 0.10 cc. octyl alcohol (different preparation).

Barometer 776 mm. He.

2 32.0 0.46 0.33 0.090 I 60

5 31.0 0.57 0.43 0.118 79

15 32.0 0.65 0.49 0.134 89

30 32.0 0.75 0.55 0.150 100

Serum globulin. 2 cc. used with 0.15 cc. oct,yl alcohol. Barometer 762 mm. Hg.

2 32 0.66 0.51 0.137 67

4 33 0.80 0.64 0.170 83

10 33 0.94 0.77 0.205 100

Crystalline egg albumin. 2 CC. used with 0.15 cc. octyl alcohol. Barometer 760 mm. Hg.

2 27.6 0.38 0.063 61

4 27.0 0.49 0.091 87

10 27.8 0.55 0.104 100

* Single determination.

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200 Amino Nitrogen in Proteins

Time. Temperature / Reading. N&-N per cc.

Purified egg albumin. 2 cc. used with 0.15 cc. octyl alcohol. Barometer 758 mm. Hg.

min. “C. cc. cc. m!J. per cent

2i 26.5 0.43 0.28 0.077 53 5 26.8 0.55 0.39 0.107 74

13 27.0 0.70 0.53 0.145 100

Edestin. 2 cc. used with 0.10 cc. octyl alcohol. Barometer 764 mm. Hg.

3 24 0.27 0.15 0.042 71 6 24 0.29 0.16 0.045 76

15 24 0.35 0.21 I 0.059 ~ 100

Bence-Jones Protein 5. 2 cc. used with 0.10 cc. octyl alcohol. Barometer 762 mm. Hg.

The method of Van Slyke seems, therefore, to be subject to error in two directions when used in analyzing protein material: (1) by yielding results which are too high, due probably to hydrol- ysis; and (2) by yielding results which are too low with certain proteins which are too insoluble in the nitrous acid. Less objec- tion can be raised to the use of Sorensen’s method with the pro- teins studied. No precipitate is formed to render the solution difficult to handle, and the titration can be carried out without difficulty even with colored solutions.

SUMMARY AND CONCLUSIONS.

The methods of Sorensen and of Van Slyke for the determina- tions of free amino nitrogen have been compared, using solutions of native and derived proteins.

The method of Van Slyke yielded results which varied depend- ing on the length of time of reaction and the material examined. Hydrolysis may account for high results and precipitation of the protein material by the reagent may account for some low results.

The method of Sorensen seems to bc less susceptible to error in the analysis of proteins.

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II. Wright Wilson 201

BIBLIOGRAPHY.

1. Van Slykc, D. D., J. l3iol. C&m., 1911, ix, 185.

2. Slirensen, S. P. L., Biochem. Z., 1908, vii, 45. 3. Van Slykc, D. D., and B&hard, F. J., J. Biol. Chem., 1913-14, xvi, 539. 4. \Yhite, G. F., and Thomas, A., J. Biol. Chem., 1912-13, xiii, 111. 5. Rogoziliski, F., Z. ph!ysiol. Chem., 1912, Ixxix, 398.

6. Andersen, A. C., Biochem. Z., 1915, lxx, 344.

7. Kossel, A., and Cameron, A. T., Z. pkusiol. Chem., 1911-12, lxxvi, 457.

8. Iiosscl, A., and Gawrilow, N., 2. $qsioZ. Chem., 1912, lxxxi, 274. 9. Abderhalden, E., and Kramm, F., 2. physiol. Chem., 1912, lxxvii, 425.

10. Northrop, J. II., J. Gen. l’hysid., 1920.-21, iii, 715.

11. Rice, F. E., J. Am. Chenz. Sot., 3916, xxxvii, 1319. 12. Harding, V. J., and MacLcan, R. M., J. Biol. Chem., 1916, xxiv, 503. 13. Folin, O., J. BioZ. Chem., 1922, li, 377.

14. Van Slyke, D. D., J. Biol. C/le??a., 1913-14, xvi, 121. 15. Sure, B., and Hart, E. B., J. Bid. Chem., 1917, xxxi, 527. 16. Dunn, M. S., and Schmidt, C. L. A., J. Biol. Chem., 1922, liii, 401.

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D. Wright WilsonAMINO NITROGEN IN PROTEINSTHE DETERMINATION OF FREE

1923, 56:191-201.J. Biol. Chem. 

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