Eldon E. Rice  PROTEIN HYDROLYSATESISOLATION OF LYSINE FROM A SIMPLIFIED PROCEDURE FOR THEARTICLE:

1939, 131:1-4.J. Biol. Chem. 

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A SIMPLIFIED PROCEDURE FOR THE ISOLATION OF LYSINE FROM PROTEIN HYDROLYSATES

BY ELDON E. RICE

(From the Ilivision of Biochemistry, Noyes Laboratory of Chemistry, Uni- versity of Illinois, Urbana)

(Received for publication, July 13, 1939)

A method has been developed for the preparation of lysine from protein hydrolysates by a direct precipitation of the amino acid as the picrate. The procedure greatly reduces the time required for the isolation of lysine and eliminates the electrolysis which is an essential part of the method of Cox, King, and Berg (1929). After one or two recrystallizations the lysine picrate obtained by direct precipitation decomposes at 263-266’, which is the same as t,he decomposition point of the product isolated by the more elaborate procedure. Moreover, the yield and quality of the lysine monohydrochloride prepared by the proposed method are as high as those obtained after electrolysis. Histidine can be separated as a by-product in both procedures. The method is adaptable to hydrolysates that have been neutralized with calcium hydroxide as well as those that have been treated with baryta. The greater ease of removal of t,he calcium sulfate by filtration, as well as the low cost of calcium hydroxide, makes this the more practical way of preparing a neutral hydrolysate if large quanbities of lysine are needed.

Triplicate lots of blood corpuscle paste have been used for the preparation of lysine monohydrochloride by the method of Cox, King, and Berg (1929), and by two modifications of the direct procedure. The results are outlined below.

EXPERIMENTAL

Three 4 liter (4.4 kilos) lots of blood corpuscle paste were re- fluxed for 24 hours, each with 4 liters of 50 per cent (by volume) sulfuric acid. The sulfate ions in two of these hydrolysates were

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2 Isolation of Lysine

removed with exactly the requisite quantity of barium hydroxide, as in the procedure of Berg and Rose (1929), and the filtrate was concentrated in vacua to 3 liters. The insoluble amino acids which separated during the concentration were removed by filtration. The syrupy liquid remaining, though free of barium ions, was defi- nitely alkaline to litmus. It was divided into two equal parts, hereafter referred to as Lot A and Lot B.

The third hydrolysate was diluted to 17 liters, and neutralized by the addition of 2700 gm. of calcium hydroxide suspended in 8 liters of water. The filtrate and washings from the calcium sul- fate were concentrated in vacua to a volume of 8 liters. Calcium hydroxide was then carefully added until the solution was alkaline to litmus. After the solution was filtered again, the hydrolysate was concentrated in vacua to a volume of 1500 cc., and allowed to stand overnight in an ice box. The insoluble amino acids which separated were removed by filtration. The syrup which remained will be referred to hereafter as Lot C.

From Lot A lysine dihydrochloride was isolated and recrystal- lized exactly as described by Cox, King, and Berg (1929). This product was dissolved in the minimum volume of boiling alcohol and converted into lysine monohydrochloride by treatment with pyridine in absolute alcohol (45 gm. of pyridine for each 100 gm. of lysine dihydrochloride). After standing overnight in an ice box, the crude monohydrochloride was collected on a Buchner funnel, and washed with a little absolute alcohol, and then with ether. Purification was accomplished by slowly adding 3 volumes of alcohol to a solution of the material in an equal weight of water. The alcohol must be added very carefully in order to promote crystallization. On the other hand, unless the addition is complete within about 10 minutes, some lysine monohydro- chloride dihydrate precipitates.

After refrigeration for 12 hours the final product was collected on a filter, washed with absolute alcohol and ether, and then dried for 4 hours at 50’ in a vacuum oven. The yield of lysine monohydrochloride was 54.0 gm.

CsH,4N202~HC1. Calculated, N 15.33; found, N 15.30

Lot B was treated directly with 150 gm. of solid commercial picric acid, and heated with frequent shaking on a steam cone

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E. E. Rice 3

at 50’ until all of the solid had dissolved. The lysine picrate which precipitated as the solution cooled (12 hours in a refrigera- tor) was collected on a 20 cm. Buchner funnel and washed twice with 200 cc. portions of cold water. The mother liquor was set aside for the preparation of histidine.’ A solution of the precipi- tate in 2500 cc. of water was boiled for 5 minutes with 70 gm. of norit and then filtered through a hot Buchner funnel. As the filtrate cooled, a solid mass of bright yellow lysine picrate formed. Since the decomposition point of this material was 261”, the prod- uct was used in the next step without further purification. In other runs the picrate obtained at this point decomposed at 255- 260’, but an additional recrystallization invariably yielded a pic- rate with the correct decomposition point (265-266”). The lysine picrate obtained from this lot was converted into the mono- hydrochloride in the manner previously described. The yield was 57.4 gm.

C~HUN~O~.HCI. Calculated, N 15.33; found, N 15.42

Lot C, which had been neutralized with calcium hydroxide, was treated in the same manner as Lot B. It yielded 55.2 gm. of lysine monohydrochloride.

CsHr,NzOz.HCl. Calculated, N 15.33; found, N 15.26

Two similar series of isolations, comparing results by the three methods, were carried out. These runs agreed closely in all re- spects with those reported above. The average yield of pure lysine monohydrochloride obtained by each of the different procedures was by the method of c/ox, King, and Berg 58.4 gm., by the direct method (neutralization with baryta) 57.9 gm., by the direct method (neutralization with calcium hydroxide) 55.6 gm.

Each sample of the lysine monohydrochloride was analyzed in duplicate by the Van Slyke procedure, and in no case did the amount of nitrogen found differ from the theoretical by more than 0.10 per cent. Each of the products gave negative Sakaguchi

1 2 kilos of sodium chloride (Gilson, 1938) were added to this solution and the histidine-mercury complex was then precipitated by the method of Foster and Shemin (1938). This material was converted into histidine dihydrochloride by the procedure outlined by Hanke and Koessler (1920). The yield was 81.9 gm. of analytically .pure product.

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4 Isolation of Lysine

tests for arginine (Jorpes, 1932). The samples of lysine mono- hydrochloride prepared from twice recrystallized lysine picrate gave negative tests for histidine (Hunter, 1922). On the other hand, lysine monohydrochloride prepared from picrate that had not been recrystallized twice gave positive tests by the Hunter method, regardless of the method of preparation. In view of this fact lysine picrate should always be recrystallized at least twice regardless of its decomposition point.

Lysine can also be prepared from dried blood fibrin by the direct method of precipitation. In demonstrating this fact, three 2.5 kilo lots of ground fibrin were hydrolyzed by refluxing for 16 hours, each with 8 liters of 25 per cent (by volume) sulfuric acid. The resulting solutions were neutralized with baryta, concen- trated, and treated with 130 gm. of picric acid per lot. The picrates were converted int,o the final product as previously de- scribed, and yielded an average of 46.5 gm. of analytically pure lysine monohydrochloride. Two additional hydrolysates which had been neutralized with calcium hydroxide yielded an average of 44.5 gm. of the pure product. 33.2 gm. of lysine monohydro- chloride were obtained from 2.5 kilos of commercial casein. An attempt to prepare lysine from gelatin was unsuccessful.

SUMMARY

A procedure for the preparation of pure lysine monohydro- chloride from protein hydrolysates by direct precipitation has been described. This method eliminates the need for special equipment, and greatly reduces the labor involved in the isola- tion of the amino acid. The yield and quality of the product are as high as those of lysine obtained by more complicated procedures. Histidine dihydrochloride may be readily prepared from the same hydrolysate.

BIBLIOGRAPHY

Berg, C. P., and Rose, IV. C., J. Biol. Chem., 82, 479 (1929). Cox, G. J., King, H., and Berg, C. P., J. Biol. Chem., 81, 755 (1929). Foster, G. L., and Shemin, D., in Fuson, R. C., et al., Organic syntheses,

New York, 18, 43 (1938). Gilson, L. E., J. Biol. Chem., 124, 281 (1938). Hanke, M. T., and Koessler, K. K., J. BioZ. Chem., 43, 521 (1920). Hunter, G., Biochem. J., 16, 637 (1922). Jorpes, E., Biochem. J., 26, 1504 (1932).

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