THE PROTEINS OF THE PEANUT, ARACHIS HYPOGJEA.

I. THE GLOBULINS ARACHIN AND CONARACHIN.

BY CARL 0. JOHNS AND D. BREESE JONES.

(From the Bureau of Chemistry, United States Department of Agriculture, Washington.)

(Received for publication, October 13, 1916 .)

The ravages of the boll weevil have made it unprofitable to grow cotton in various sections throughout the South. In many regions where the growing of cotton has been decreased, peanuts are now being grown as a supplementary crop. This has caused a large increase in the production of peanuts in the United States. A number of mills which in the past produced cottonseed oil are now using their presses for the production of peanut oil. The press-cake thus obtained as a by-product is used for cattle food and sells at about thirty-five dollars a ton. This cake, therefore, furnishes a relatively cheap source of food with a very high pro- tein content.

The most striking result brought out during this investigation is the fact ‘that the globulins of the peanut contain a high percent- age of basic nitrogen when compared with the proteins of other seeds commonly used for food. Work on the hydrolysis and separation of the amino-acids of the peanut globulins is in prog- ress and will be reported in a later publication.

The proteins of the peanut have received but little attention. The only published experiments seem to be those described by Ritthausenl in a paper which appeared in 1880. This author ex- tracted oil-free peanut meal with a solution of sodium chloride and with solutions of potassium, calcium, and barium hydroxides. From the sodium chloride extract he obtained the globulin by saturating the diluted extract with carbon dioxide, or simply by diluting the extract with a large volume of water. Apparently the same globulin was obtained by acidifying the alkaline ex-

1 Ritthausen, H., Arch. ges. Physiol., 1880, xxi, 81.

77

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Proteins of Peanut. I

tracts with acetic or sulfuric acids. Ritthausen found no evidence in his analytical data that indicated the presence of more than one globulin in the peanut.

The peanut meal with which Ritthausen experimented had been dried over sulfuric acid in a desiccator and contained 10.18 per cent of nitrogen or 56 per cent of protein. The latter value was obtained by multiplying the percentage of nitrogen by 5.5, thus assuming that the protein contained 18 per cent of nitrogen. When this meal was extracted with 10 per cent sodium chloride solution he isolated 27 per cent of globulin by diluting the extract with water and then saturating with carbon dioxide.

We have extracted air-dried oil-free peanut meal containing 42 per cent of protein (N X 5.5) with a 10 per cent sodium chlo- ride solution and have found that about 32 per cent of protein, based on the weight of meal used, is dissolved at room tempera- ture. When the temperature was raised to 40-50°C. the yield was not increased. From a sodium chloride extract of the meal, calculated to contain 32 gm. of globulin, we were able to isolate about 25 gm. by diluting the sodium chloride extract with water or by saturating with carbon dioxide. This yield is based on the actual weight of pure protein after drying at 100°C. and does not take into consideration the losses incurred during its isolation.

The analyses of ten preparations of peanut globulin precipi- tated by dilution or by dialysis of sodium chloride extracts gave results which agree well with those published by Ritthausen, except the value for carbon, which we found to be about 0.7 per cent higher than that reported by him.

We have made fractional precipitations of the protein extracted by salt solutions from peanut meal and in this manner have iso- lated two globulins, one of which is present only to a small ex- tent. The globulin composing the greater part of the precipitate obtained by diluting sodium chloride extracts of peanut meal with water is the less soluble of the two and is precipitated, when in 10 per cent salt solution, by adding ammonium sulfate to 0.2 of saturation. To this globulin we have given the name arachin. After filtering off the arachin the second globulin may be obtained by dialysis or by saturating the filtrate with ammonium sulfate. This second globulin we propose to call conarac&~ The differ-

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C. 0. Johns and D. B. Jones 79

ence in composition of these two globulins is shown by the fol- lowing analyses.

c .......................... H .......................... N .......................... s .......................... 0. .........................

.

........

........

........

Arachin. Conarachin.

52.15 51.17 6.93 6.87

18.29 18.29 . . 0.40 1.09

22.23 22.58

It will be seen that the greatest difference between these two globulins is in the percentage of sulfur, which is nearly three times as great in conarachin as in arachin Another striking dif- ference between these two proteins is shown by the figures rep- resenting the distribution of the nitrogen. The figures are as follows :

N Amohin. Conarachin.

Amide ................................... 2.03 Humin ................................... 0.22 Basic .................................... 4.96 Non-basic ................................ 11.07

2.07 0.22 6.55 9.40

Total.................................... 18.28 I

18.24

The difference in the basic nitrogen as shown by these results is most striking for, as far as we know, conarachin contains more basic nitrogen than any other seed globulin yet examined. The percentage of basic nitrogen in the mixture of these globulins is abo high, being 5.23 per cent. For the sake of comparison the percentages of basic nitrogen in some proteins from seeds com- monly used for foods are given below.2

* Osborne, T. B., The Vegetable Proteins, London, 1909, 57.

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80 Proteins of Peanut. I

Protein.

Zein ....................................... Gliadin ....................................

“ .................................... Phaseolin .................................. Vicilin ..................................... Arachin .................................... Legumin ................................... Excelsin ................................... Edestin .................................... Globulin ................................... Conarachin ................................

-

-

SOUIW.

Maize. Rye. Wheat. Kidney bean. Pea. Peanut,. Pea. Brazil nut. Hempseed. Cocoanut. Peanut.

Basic nitrogen.

per cent

0.49 0.91 1.09 3.62 4.92 4.96 5.11 5.76 5.97 6.06 6.55

Besides arachin and conarachin, sodium chloride solutions ex- tract from peanut meal a trace of albumin which coagulates at 65-70°C. The quantity of albumin is so small that we have not yet been able to obtain a pure product in sufficient amounts for a complete analysis. Carbon, hydrogen, and nitrogen determina- tions made on a small quantity of this albumin gave results which agree closely with the values obtained by Osborne and his coworkers3 for the legumelins frequently found in the seeds of leguminous plants.

If the diamino-acids represented by the basic nitrogen of the peanut proteins are present in the proper proportions, peanut press-cake may be used to supplement the deficiency of basic nitrogen in such foods as wheat, oats, and corn. Mixtures of these cereals with peanut press-cake or peanut flour may prove to be a highly nutritious diet and may be used as food for human beings as well as animals at less cost than when the cereals are used alone.

Investigations on the proteins of the peanut will be continued.

EXPERIMENTAL.

Preparation of Meal from the Peanut.-Shelled Virginia pea- nuts of the best quality were used in this investigation. The

8 Osborne, Ergebn. Physiol., 1910, x, 126.

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C. 0. Johns and D. B. Jones 81

peanuts were pressed cold in an Anderson expeller to remove most of the oil. The resulting press-cake was then ground to a meal, the residual oil was removed by means of cold petroleum ether, and after filtering by suction the meal was placed in a vacuum desiccator until free from petroleum ether.

Globulins Precipitated by Diluting Xodium Chloride Extracts of Peanut Meal with Water.-In extracting the globulins from pea- nut meal 500 cc. of a 10 per cent sodium chloride solution were used for each 100 gm. of meal. The meal was stirred into the salt solution, giving a thick pasty mass. This paste was run through a grinding mill three times and a thin mixture was ob- tained as the distended cell walls were broken up. Dry filter paper scrap was then added and the mixture was worked up with the hands until a rather stiff pulp was obtained. This was placed in cloths and the liquid forced out by means of a powerful press. In this way we pressed out a volume of extract equal to about 80 per cent of that of the sodium chloride solution used. The press juice was then filtered clear by suction through a thick mat of paper pulp and a clear fluorescent liquid was obtained. This was diluted by adding five or six volumes of distilled water. The globulins precipitated immediately in the form of a viscous paste, which, when stirred with a rod, exhibited a silvery sheen. The supernatant liquid was poured off and a little distilled water added. On stirring with a glass rod the character of the precipi- tate was altered so that it was converted into a finely divided granular substance and an opaque milk-like fluid. The latter could neither be filtered nor caused to separate by centrifugating. The addition of alcohol at this stage usually caused the milky liquid to separate so that it could be filtered off by suction or thrown down by centrifugating. The precipitate thus produced was washed with 50 per cent alcohol and then dried by suspend- ing it in absolute alcohol for 24 hours. The protein was then fil- tered off and suspended in anhydrous ether for another 24 hours. After filtering off the ether the protein was placed in a vacuum desiccator and was finally dried in a vacuum oven by raising the temperature gradually to 110°C. The globulin was thus obtained as a white powder. Analyses of five different preparations are given below.

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82 Proteins of Peanut. I

c ............................... H ............................... N ............................... s ............................... 0 ............................... Ash. ............................

I I Preparation 1. Preparation 2.

-~- --- 51.48 52.11 51.48 51.35 51.41 52.06

6.61 6.69 6.63 6.63 6.71 18.01 18.22 18.05 18.05 18.27

0.46 0.46 0.42 0.42 0.43 22.52 22.53

1.18 1.22 1.22

Preparation 3. Preparation 4.

I. II. Aver- B&3. k? k%

~--__~-

C . . . . . . . . . . . . ..___..__.__........ 51.42 51.52 51.47 51.87 52.10 52.53 H . . . . . . . . . . . . . . . . . . . ..__...__. 6.76 6.70 6.73 6.78 6.74 6.79 N . . . . . . . . . . . . . 18.19 18.23 18.21 18.34 18.17 18.32 S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.48 0.48 0.48 0.61 0.61 0. . . . . . . . . . . . 22.53 21.75 Ash . . . . . . . . . . . . 0.78 0.78 0.80

Preparation 5.

I. II. AWX- Ash- age. free.

~-~-

C . . . . . . . . . . . . . . .._......_______..._......_..._ 51.54 51.78 51.66 52.11 H . . . . . . .._..........._.__.._....._ ,....._._... 6.65 6.74 6.69 6.72 N . . . . . . . . . . . . . . . . . . . . . . .._.__._..._.____.___.. 17.87 17.87 18.03 9 L............................................. 0.41 0.41 0.41 0. . . . . . 22.73 Ash.. . . . 0 87 0.87

Globulins Obtained by Dialysis of Sodium Chloride Extracts of Peanut Meal.-Sodium chloride extracts of peanut meal were pre- pared as described above. These extracts were dialyzed in parch- ment bags, suspended in running water, until the solution ceased to give a test for chlorine. Toluene was used to keep the solu- tion sterile. The analyses of three preparations thus obtained are given below. It will be seen that these preparations differ but slightly from those obtained by precipitating the globulins by adding water to the sodium chloride extract.

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C. 0. Johns and D. B. Jones 83

Preparation 6. Preparation 7. Preparation 8.

I. II. Aver- Ash- free. I. II. Aver- Ash- Aver- Ash-

age. age. free. 1. II. age. free. __-___

C . . . . . 51.9152.1152.0152.3952.0452.1252.0852.4051.3751.3251.3451.60 H..... 6.57 6.76 6.66 6.71 6.67 6.75 6.71 6.75 6.68 6.64 S.fjS 6.69 N . . . . . 18.0118.1018.0518.2417.7717.8717.8217.9217.95 17.9518.05 s..... 0.50 0.50 0.50 0.48 0.48 0.48 0.67 0.62 0.64 0.64 O..... 22.16 22.45 23.02 Ash... 0.76 0.76 0.59 0.59 0.55 0.55

Globulins Obtained by Saturating Sodium Chloride Extracts of Peanut Meal with Carbon Dioxide.-Sodium chloride extracts of peanut meal were diluted with water until a faint turbidity was produced and were then saturated by passing a stream of carbon dioxide through the liquid. A compact white precipitate was obtained on allowing the mixture to settle over night. Two preparations made in this way gave the results stated below.

Preparation 9. Preparation 10

I. II. ATW- Ash- I, AWX- IL zL-3.

Ash- age. free. .a free.

~__ ~-

C . . . . . . . ..__........ 52.17 52.85 52.01 52.17 52 21 52.00 52.10 52.29 H . . . . . . . . . ..__.__._. 6.81 6.75 6.78 6.79 6.51 6.71 6.61 6.63 N . . . . . . . . . .._..__ 18.17 18.21 18.19 18.24 18.08 18.08 18.15 s . . . . . . . . . . . . .._ 0.49 0.49 0.49 0.42 0.42 0.42 0 . 22.31 22.51 Ash . _. 0.29 0.29 0.34 0.34

Summary of Analyses of the Total Globulin Obtained from the Peanut.

Preparation No.. 1. 2. 3. 4. 5. 6. ~-

C . . . . .._...............___.._.._. 52.11 52.06 51.87 52.53 52.11 52.39 H................................ 6.69 6.71 6.78 6.79 6.72 6.71 N................................ 18.22 18.27 18.34 18.32 18.03 18.24 S .._.__............__.,.....,,,,. 0.46 0.43 0.48 0.61 0.41 0.50 0 . . . . . . . . . . . . . . ..____...._... 22.52 22.53 22.53 21.75 22.73 22.16

~~ __- Preparation No.. . 7. 8. 9. 10. Aver-G-

age. hausen. -~-~

C... . . . . . . . . . ..__._..._..... 52.40 51.60 52.17 52.29 52.15 51.41 H . . . . . . . . . . . . . . . . . . . . . . . .._..._.. 6.75 6.69 6.79 6.63 6.72 6.74 N................................ 17.92 18.05 18.24 18 15 18.18 18.25 S . . . . . . . . . . . . . . . . . . . . . .._.._..... 0.48 0.64 0.49 0.42 0.50 0.56 0 . . . . .._....____........,......,. 22.45 23.02 22.31 22.51 22.45 23.04

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Proteins of Peanut. I

Nitrogen Distribution in the Total Globulin in the Peanut.--The diskibution of the nitrogen in the total globulin obtained from the peanut was found by Hausmann’s method as modified by Osborne and Harris. Results obtained on two different prepa- rations (a and b) are given below. The calculations were made on an ash- and moisture-free basis.

N

Amide. Humin.......................... Basic........................... Non-basic.

Total...........................

Preparation a. Preparation b.

___~~__ ~~ 2.05 2.10 2.08 2.07 2.09 2.08 0.18 0.24 0.21 0.21 0.19 0.20 5.29 5.22 5.25 5.18 5.25 5.21

10.63 10.59 10.61 10.78 10.71 10.75 ~~ -__ ~-

18.15 18.15 18.15 18.24 18.24 18.24

Fractional Precipitation of the Globulins of the Peanut.

Isolation of Arachin.-An extract was made as previously de- scribed, using 500 gm. of peanut meal and 2.5 liters of a 10 per cent sodium chloride solution. To the clear filtrate solid ammo- nium sulfate was added gradually until the solution became 0.2 saturated with this salt. A precipitate began to appear at 0.15 of saturation and increased rapidly until 0.2 of saturation was reached, when precipitation practically ceased and did not occur again until much more ammonium sulfate was added. On al- lowing the 0.2 saturated solution to stand over night, the precipi- tate settled out in a very compact form so that the supernatant liquid could be easily decanted. The precipitate was washed with a 10 per cent sodium chloride solution containing ammonium sulfate to 0.2 of saturation. The residue was then redissolved in a small volume of 10 per cent sodium chloride, and the result- ing solution was filtered and dialyzed until chlorides were re- moved. In this manner the greater part of the globulin present in the meal was obtained as a white powder. This was dried for analysis in the manner previously described. Three different preparations gave the following results.

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Preparation 11. Preparation 12. Preparation 13.

I. II. Aver- $;; age.

I . 11. age.

Aver- Ash- I. free.

II Aver- fr;s; . age.

-~~-_--- ____--

C . 52.0852.2552.1652.5151.5551.5351.5451.8651.9051.8451.8752.16 H . . . . . 6.96 6.98 6.97 7.02 6.96 6.81 6.88 6.93 6.95 6.80 6.87 6.91 N . . . . . 18.1618.1218.1418.2618.2218.2718.2418.3718.17 18.1718.28 s..... 0.37 0.37 0.37 0.41 0.41 0.41 0.44 0.44 0.44 o.... 21.84 22.43 22.21 Ash.. 0.69 0.69 0.62 0.62 0.57 0.57 -

Distribution of the Nitrogen in Arachin.-The sample used for this experiment contained 18.28 per cent of nitrogen, calculated on an ash- and moisture-free basis. The results are given below.

N

Amide ............................. Humin ............................ Basic .............................. Non-basic .........................

Total .............................

I.

2.06 0.23

II. AXWage.

2.00 2.03 0.20 0.22 4.96 4.96

11.12 11.07

18.28 18.25

Isolation of Conarachin.-The filtrate from Preparation 12, from which arachin had been removed, as described above, and which was now 0.2 saturated with ammonium sulfate, was com- pletely saturated by adding more ammonium sulfate. This treat- ment produced a small quantity of precipitate. This was redis- solved in 10 per cent sodium chloride and the filtered solution dialyzed until free from chlorides. A small quantity of con- arachin, Preparation 14, was obtained. Another preparation of conarachin was made by dialyzing the filtrate from No. 13. This was designated No. 15. These preparations gave the following results on analysis.

Preparation 14. Preparation 15.

I. II. Aver- AVET- Ash- age. Ez I. II. age. free.

---

C . . . . . .._.._........ 50.89 51.12 51.00 51.16 50.95 51.03 50.99 51.19 II . . . . . .._._......... 6.86 6.82 6.84 6.87 6.77 6.89 6.83 6.S6 N . . .._.__._.____.._. 18.34 18.24 18.29 18.34 18.13 18.22 18.17 18 24 S . . ..._.___...,..,., 1.07 1.05 1.06 1.06 1.12 1.12 1.12

0 . . . . 22.57 22.59 Ash................. 0.32 0.32 0.37 0.37

85

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86 Proteins of Peanut. I

Distribution of the Nitrogen in Conarachin.-The sample used contained 18.24 per cent of nitrogen.

N

Amide ............................. Humin ............................ Basic .............................. Non-basic. ........................

Total ..............................

I. II. Average.

2.07 2.06 2.07 0.23 0.21 0.22 6.59 6.51 6.55 9.35 9.46 9.40

18.24 18.24 18.24

Comparison of Analyses of Aro.chin and Conarachin.

c .............................................. H .............................................. N .............................................. s .............................................. 0. .............................................

Araohin, average of

Conarachin,

Preparations rtverage of

Preparations 11.12, and 13. 14 and 15.

52.15 51.17 6.93 6.87

18.29 18.29 0.40 1.09

22.23 22.58

Comparison of the Nitrogen Distribution in the Total Globulins an2 in Arachin and Conarachin.

N Total globulins. Arachin. Conaraohin.

Amide. . . . . . . . . . . . . 2.08 2.03 2.07 Humin............................ 0.21 0.22 0.22 Basic.............................. 5.23 4.96 6.55 Non-basic.. . . . . . . . . . . . . 10.68 11.07 9.40

Total.............................. 18.20 18.28 18.24

SUMMARY.

1. Two globulins have been isolated from the peanut. These have been named arachin and conarachin.

2. Arachin contains 0.4, and conarachin 1.09 per cent of sulfur. 3. Arachin gave 4.96, and conarachin 6.55 per cent of basic

nitrogen, the latter being the highest percentage of basic nitrogen recorded for any seed protein.

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C. 0. Johns and D. B. Jones 87

4. From the results that we have obtained it seems probable that peanut press-cake will prove to be highly effective in sup- plementing food products made from cereals and other seeds whose proteins are deficient jn the basic amino-acids. Feeding experi- ments are already in progress to determine the nutritive value of combinations of peanut proteins with other proteins obtained from the more extensively used seeds.

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Carl O. Johns and D. Breese JonesCONARACHIN

GLOBULINS ARACHIN ANDARACHIS HYPOGÆA: I. THE

THE PROTEINS OF THE PEANUT,

1916, 28:77-87.J. Biol. Chem. 

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