PROTEINS OF THE BLACK BEAN OF THE MAYAS, PHASEOLUS VULGARIS

BY D. BREESE JONES, CHARLES E. F. GERSDORFF, AND SAMMIE PHILLIPS

(From the Protein and Nutrition Research Division, Bureau of Chemistry and Soils, United States Department of Agriculture, Washington)

(Received for publication, November 10, 1937)

There came recently to our hands a sample of black beans which Dr. S. G. Morley of the Smithsonian Institution had collected in Yucatan, where they are used as the principal supplement to maize in the diet of the people. The Mayas, he reported, subsist al- most entirely on maize and beans, and are a sturdy, healthy race. A bush bean probably identical with the beans Dr. Morley col- lected in Yucatan is grown in South America and used extensively for food. It is there referred to as the black bean, and its history dates back to olden days.l

Dr. V. R. Boswell of the Bureau of Plant Industry kindly iden- tified the sample referred to as Phaseolus vulgar&, and advised that they are known as the “black turtle soup” bean. They are apparently identical in type with a large number of specimens of black turtle soup bean introduced from Guatemala about 15 years ago and are similar in properties to the kidney bean. Although there is not much trade in them in the United States, they are on the market to some extent but are not popular because they are a black bean.

A quantity of the beans sufficient for a study of their proteins was received from the Division of Plant Exploration and Introduc- tion through the kind offices of Dr. F. D. Richey of the Bureau of Plant Industry. These beans were shipped from Costa Rica and were used for the work described in this paper.

In view of the reported extensive use of these beans together

1 Personal communication from Dr. W. E. Whitehouse, Bureau of Plant Industry, United States Department of Agriculture, Washington.

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746 Proteins of Black Bean

with maize in the diet of the Mayas, it was considered of interest to study the possible supplementary relationship that may exist between their proteins and those of maize with respect to amino acid composition, and to compare their properties and composi- tion with those of other varieties of beans which have been studied.

Most legume seeds contain two types of globulins which have been designated a-globulins and p-globulins (1). These globulins are differentiated primarily by their solubilities, coagulation tem- peratures, and amino acid composition. Strikingly characteristic are their differences in cystine content. The cystine content of the a-globulins ranges from 7 to 8 times that of the fl-globulins. Usually the p-globulins constitute by far the greater part of the total globulin of the seeds.

It is seldom that a definite concentration of ammonium sulfate can be used which will effect a sharp separation of the two glob- ulins. Generally a middle fraction from the apparent upper limit of the first main precipitate to the apparent lower limit of the second is removed before the latter is precipitated. This middle fraction is generally found to be a mixture of the two globulins.

Preliminary tests with NaCl solutions showed that the maxi- mum amount of nitrogen was removed by a 2 per cent solution of this salt. 5 cc. of the solvent per gm. of meal were used, and the extraction was carried on for 2 hours with stirring. Repeated extractions removed 82 per cent of the total nitrogen of the meal. Most of the a-globulin was precipitated from the extract at 35 per cent of saturation with ammonium sulfate. The fraction that further separated up to 55 per cent of saturation consisted of a mixture of the (Y- and /3-globulins. Increasing concentrat,ion of ammonium sulfate up to 80 per cent of saturation gave a heavy precipitate of the fi-globulin. Additional ammonium sulfate caused no further precipitation.

Conclusive evidence of the presence of albumin or proteose in the extract was not found in the preliminary tests or in the sub- sequent part of the study.

Preparation of Proteins

The beans were ground to a fine meal in a power-driven mill. Care was taken to avoid heating. The meal contained 3.81 per cent nitrogen.

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2 kilos of freshly ground meal were extracted with 10 liters of 2 per cent NaCl solution for 2 hours at room temperature with frequent stirring. The mixture was centrifuged, and the residue was washed once with 2 per cent NaCl solution. The clear, dark purplish extract had a reaction of pH 7.0. The combined extract and washing measured 11 liters.

Lu-Globulin-The a-globulin was precipitated from the clear extract of the meal by addition of 26.7 gm. of ammonium sulfate per 100 cc. of extract (35 per cent of saturation). The globulin was separated by centrifugation and was washed five times with 2 per cent NaCl solution containing the same concentration of ammonium sulfate as that from which it was precipitated. The combined centrifugates were reserved for the preparation of the p-globulin. The washed cr-globulin was dispersed in 2 liters of 2 per cent NaCl solution and the filtered solution was dialyzed for 18 days against running cold water. Toluene was used as pre- servative. The globulin which precipitated was washed and dried with alcohol and ether2 in the usual way (Preparation I). The preparation weighed 4 gm. and consisted of a dark, drab powder.

The highly opalescent dialysate from the a-globulin had a reac- tion of pH 7.1. It became turbid but yielded no coagulum when slowly heated at 84-90”. Addition of a small amount of acetic acid after it reached this temperature, enough to bring the solu- tion to pH 6.8, caused the separation of a heavy coagulum. Strangely, however, when the solution was adjusted to pH 6.8 before heating, no coagulum formed on subsequent heating. The coagulum was washed and dried in the usual manner (Prepara- tion II). It consisted of a brownish drab powder,3 and weighed 6 gm. Analysis of this preparation showed it to be practically identical in composition with Preparation I, indicating that it was a slightly changed form of the ac-globulin, a change probably brought about during the dialysis by enzymatic action.

2 Evaporation of the ether washings yielded a small quantity of a heavy, oily substance. This was examined by Dr. R. S. McKinney of the Oil, Fat and Wax Laboratory of this Bureau. It was found to have a saponification value of 73.3. It proved not to be a fat, and gave negative tests for carbo- hydrate and saturated hydrocarbon. It was believed to be of a lipid nature.

3 The color of the o(- and p-globulin preparations was due to the absorp- tion of a trace of pigment extracted from the black hulls of thebea,ns. It had no significant effect upon their composition.

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748 Proteins of Black Bean

The combined weight of Preparations I and II amounted to 10 gm.

/3-Globulin-The intermediate fraction of mixed globulins was removed from the filtrate remaining after separation of the a- globulin by further addition of 15.26 gm. of ammonium sulfate per 100 cc. of the filtrate (enough to bring it up to 55 per cent of saturation). The precipitate was removed by filtration on a pad of filter paper pulp which had been moistened with a 55 per cent saturated solution of ammonium sulfate.

There were then added to the filtrate from the middle fraction 19.08 gm. of ammonium sulfate per 100 cc. to bring it up to 80 per cent saturation. The precipitated p-globulin was filtered off on folded filter papers. The globulin was then dispersed in 2 liters of distilled water. The amount of ammonium sulfate adher- ing to the moist precipitate was sufficient to effect solution with- out the use of additional salt. The solution was filtered and was dialyzed for 19 days. The precipitated protein was washed, and dried in the usual manner with alcohol and ether. The product consisted of a light, cream-colored powder, which weighed 62 gm. (Preparation III).

This amount of material did not account for nearly all of the p-globulin which had been precipitated by ammonium sulfate. The dialysate was, therefore, carefully examined for other protein material. When heated to 65-68’ a coagulum separated. Analy- sis of the washed and dried coagulum (Preparation IV) gave re- sults closely agreeing with those obtained for Preparation III. This preparation doubtless represents a slightly changed form of the P-globulin produced as the result of enzymatic action during the rather long period of dialysis to which it had been subjected. This product weighed 18 gm., which together with Preparation III amounted to 80 gm. of the p-globulin.

Properties of Globulins

or-Globulin-The a-globulin of the black bean is readily dis- persed in 2 per cent NaCl solution, from which it is precipitable by addition of sufficient ammonium sulfate to make the solution 35 per cent saturated with this salt. It cannot be reprecipitated from the salt solution at room temperature by dilution with 20 volumes

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of water. It precipitates from the diluted solution, however, after standing for several hours at 10”. It coagulates at 75” in a 2 per cent NaCl solution acidified with acetic acid to pH 6.8. A neutral salt solution of the globulin, however, becomes only densely turbid at 90” but does not flocculate.

Like the cY-globulins of other beans of the Phaseolus genus, the a-globulin of the black bean is characterized by a lower nitrogen content, and a much higher cystine and tryptophane content, than that of the p-globulin.

The dried a-globulin was obtained as a brownish drab powder. /%GZobuZi?b-The p-globulin readily disperses in 2 per cent NaCl

solution, and can be reprecipitated by addition of ammonium sulfate to 80 per cent of saturation. When the NaCl solution of the globulin is diluted with 20 volumes of water at room tempera- ture, the mixture becomes milky. The protein precipitates after standing for several hours in a refrigerator. Without acidifica- tion it only partially coagulates in 2 per cent NaCl solution at 90°, but it is completely precipitated at this temperature after the solution has been adjusted to a reaction of pH 6.8 by addition of acetic acid.

The dried preparation as obtained consisted of a light cream- colored powder.

Analyses of Protein Preparations

The nitrogen, sulfur, ash, and moisture contents of the cy- and p-globulin preparations are given in Table I. The distribution of nitrogen in the proteins as determined by the Van Slyke method (2) is shown in Table II.

In Table III are given the percentages of some of the amino acids found in the proteins. Cystine was determined by the method of Sullivan (3), tryptophane by the method of May and Rose (4), and tyrosine according to the method of Folin and Ciocalteu (5). The figures for arginine, histidine, and lysine were calculated from the results of the Van Slyke analyses given in Table II.

The percentages given in Tables I to IV have been calculated on the basis of ash- and moisture-free protein.

For comparison of the data found for the proteins of the black bean with those similarly obtained for other beans of the same

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genus, there are given in Table IV figures previously determined on the proteins of the navy bean (l), mung bean (6), ads&i bean (7), and the Lima bean (8).

TABLE I

Elementary Composition of LY- and p-Globulins of Black Bean

I a-Globulin I @-Globulin

Nitrogen*. ...................... Sulfur* .......................... Ash. ............................ Moisture. .......................

15.60 1.03 0.61

10.21 -

Prepara- Prepara- tion II tion III

per cent per cent 15.71 16.57

1.03 0.27 0.35 1.24 8.65 8.27

Prepara- tion IV

per cent 16.62

0.28 0.75 9.53

* Percentages calculated on the basis of ash- and moisture-free material.

TABLE II

Distribution of Nitrogen in LX- and B-Globulins

AmideN ........................ Humin “ adsorbed by lime .......

‘I IL in ether-amyl alcohol- extract ........................

ArginineN ....................... Cystine N ....................... Histidine N ..................... Lysine N ........................ Amino “ of filtrate. ............. Non-amino N of filtrate ..........

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

J

-.

ol-Globulin* &Globulin

Preparation II

per cent 9.86

3.03

‘reparation III il! ?reparation IV

per cent 10.98

2.78

per cent

10.72 2.67

0.42 0.53 0.45 10.96 12.12 12.07

0.91 0.89 0.85 4.41 5.47 5.39 7.81 7.76 7.78

60.97 56.07 57.06 2.08 3.04 2.62

100.45

_

93.64 99.61

* Because of insufficient material the distribution of nitrogen was not determined on Preparation I.

In general, there is quite a close agreement in composition be- tween the corresponding proteins of the different beans. This is particularly true with respect to their nitrogen and sulfur con- tent. Tvrosine and lvsine are nresent in relatively large amounts

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Jones, Gersdorff, and Phillips 751

in both globulins of all the beans. The ac- and p-globulins of the black bean, however, contain appreciably less lysine than the cor- responding globulins of the other beans, with the exception of

TABLE III

Amino Acids in a- and @-Globulin Preparations

Amino acid 1 o-Globulin I I- - P;@-oP$=Ia-

per cent

Arginine. ......................... Histidine ......................... Lysine ............................ Cystine ........................... 0.77 Tryptophane ..................... 2.94 Tyrosine .......................... 5.05

PR?pk?&% tion III

pet cent per cent pm cent per cent 5.34 6.24 6.23 1.8 2.56 3.34 3.30 0.8 6.39 6.71 6.75 0.0 0.77 0.11 0.12 0.8 3.04 0.98 0.99 0.17 5.10 4.25 4.27 5.9

@-Globulin Zsin

TABLE IV

Comparison of Globulins of Black Bean with Those of Other Beans of Phaseolus Group

The values are expressed as percentages of the proteins.

rr-Globulins @-Globulins

Black turtle Navy Mung Ad- Black

so”,“,“, bean bean Et; Lima turtle Navy Mung ,“,: i:;; bean soo”, bean bean bean

~-~ ---

Pu’itrogen........15.7115.6515.6715.6015.5516.5716.3016.8316.5714.81 Sulfur _,._,_.._.. 1.03 1.38 1.50 1.21 1.27 0.27 0.33 0.41 0.40 0.35 Cystine*.. .._. 0.77 0.79 1.37 0.51 1.15 0.11 0.12 0.06 0.13 0.12 Arginine.. 5.34 6.87 5.13 5.45 5.67 6.24 6.36 7.56 7.00 5.07 Histidine........ 2.56 0.85 3.30 2.25 3.71 3.34 2.36 2.02 2.51 2.62 Lysine.......... 6.3910.69 6.08 8.30 7.84 6.71 9.42 9.29 8.41 8.53 Tryptophane.... 3.04 2.79 2.03 1.72 1.92 0.98 0.94 1.18 0.96 2.16 Tyrosine........ 5.10 4.95 3.70 4.28 4.97 4.25 4.16 4.84 3.91 4.27

* All the cystine determinations were made calorimetrically by the method of Sullivan (3).

that for the a-globulin of the mung bean. The p-globulins of all the beans are characteristically low in cystine. The histidine content of the a-globulin of the black bean is 3 times that of the corresponding globulin of the navy bean, but about one-third

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752 Proteins of Black Bean

less than that of the Lima and mung beans. The p-globulin of the black bean, however, contains about one-third more histidine than the /I-globulins of the other beans. Since the /3-globulins constitute by far the greater proportion of the total protein of the seeds, it would appear that the black bean is a considerably better source of histidine than the other beans which have been consid- ered. The tryptophane value of the total globulins of the black bean closely approximates that of the other beans.

The biological value of a protein depends not only upon its absolute content of the dietary essential amino acids, but also on the extent to which they can be utilized by the body for nutri- tional requirements. Digestion studies, both in vitro and in wivo, have showed that the proteins of practically all the beans of the Phaseolus genus have a low coefficient of digestibility when in the raw state. Rats will not grow satisfactorily when raw navy bean meal, for example, or its isolated proteins constitute the sole source of protein in an otherwise adequate diet, even after supple- mentation with cystine, unless the proteins have been heated sufficiently to coagulate them. Although no data are available on the digestibility of the black bean proteins, the fact that its proteins agree so closely in chemical composition with those of other beans botanically related makes it appear very probable that they also share with them the property of indigestibility when in the raw state.

The large extent to which the diet of the Mayas consists of maize and the black bean lends interest to a comparison of the amino acid content of the proteins of these two seeds from a con- sideration of the possible supplementary value of their proteins. To what extent do the proteins from the one source supply a lib- eral amount of the dietary essential amino acids which are lacking or deficient in the other?

Of the amino acids determined in the proteins of the black bean, lysine, histidine, and tryptophane are generally recognized as dietary essential amino acids. Until recently, cystine has also been regarded as dietary essential. Jackson and Block (9) and Rose et al. (10) have shown, however, that cystine can be replaced in the diet by methionine. Little, if any, information is available on the methionine content of the proteins of beans. Whatever

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Jones, Gersdorff, and Phillips 753

may be the relationship between these two amino acids in the nutritional properties of the proteins of legume seeds, it has been amply demonstrated by different workers that cystine is a limiting factor.

Rose and coworkers have recently added several other amino acids to the list of those which are dietary essential. These in- clude leucine, isoleucine, phenylalanine, threonine, and valine. In considering the supplementary relationship between different proteins, the percentages of these amino acids should also be com- pared, but at present this is not practical because sufficiently accurate data on the amounts present in proteins are not available.

In comparing the proteins of the black bean with those of maize from the standpoint of supplemental values, emphasis should be laid on the relative amounts of lysine, cystine, and tryptophane present, for these amino acids are known to be limit- ing factors in the proteins of one or the other of these seeds. In Table III are given for comparison figures showing the percentages of amino acids in zein, the chief protein of maize.

The lysine content of the proteins of the black bean like that of most legume seeds exceeds that of most food proteins. Zein is devoid of this amino acid. The proteins of the black bean have practically the same low content of cystine as the corresponding proteins of the navy bean, which are inadequate for supporting growth unless supplemented with cystine. The cystine content of zein is a little higher than that of the a-globulin of the black bean and about 7 times that of the P-globulin, which is the pre- dominating protein of the bean. There is no evidence, as far as we are aware, that cystine is a limiting factor in the total proteins of maize.

Zein is conspicuously lacking in tryptophane. There is a fair amount of tryptophane in the P-globulin of the black bean, and more in the a-globulin than in most proteins. The amount of histidine in zein is also much lower than in the black bean pro- teins.

It is realized that the above comparisons are made between two globulins of the black bean which comprise the greater part of its total protein content, and zein which represents only one of the maize proteins. Other proteins occur in maize in smaller pro-

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754 Proteins of Black Bean

portions, but not much is known concerning their amino acid com- position. However, it is believed that the total proteins of maize have the same amino acid deficiencies as zein, although to a lesser degree. Hogan (11) has shown by feeding experiments that the total proteins of maize do not supply enough lysine or tryptophane to meet the growth requirements of the rat and that tryptophane is the first limiting factor.

On the basis of the experimental data presented it is not be- lieved that a significant superiority in nutritive value can justly be claimed for the proteins of the black bean over those of most of the other beans which have been considered. In general, there is a striking similarity between the different varieties of the Phaseolus beans with respect to the chemical and physical proper- ties of their proteins. It would seem that a supplemental re- lationship exists between the proteins of maize and those of all these beans. The relatively higher content of histidine in the black bean proteins may, however, entitle them to the claim of some preference.

SUMMARY

The chief proteins of the black turtle soup bean, a variety of Phaseolus vulgaris, consist of an CY- and a p-globulin. These globulins have been isolated and a study made of their nitrogen distribution and amino acid content. They are strikingly similar in their chemical and physical properties to the corresponding proteins of other beans of the Phaseolus genus.

It is reported that the Mayas subsist almost entirely on maize and this bean, and are a sturdy, healthy race. A comparison of the amino acid composition of the black bean proteins with those of maize indicates a supplemental relationship. The relatively large amount of lysine, tryptophane, histidine, and cystine in the bean proteins is in marked contrast to that of maize.

BIBLIOGRAPHY

1. Waterman, H. C., Johns, C. O., and Jones, D. B., J. Bid. Chem., 66, 93 (1923).

2. Van Slyke, D. D., J. Biol. Chem., 10, 15 (1911-12); 22, 281 (1915). 3. Sullivan, M. X., Pub. Health Rep., U. S. P. H. S., suppl. 78 (1929). 4. May, C. E., and Rose, E. R., J. Biol. Chem., 64,213 (1922).

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5. Folin, O., and Ciocalteu, V., J. Biol. Chem., 73,627 (1927). 6. Johns, C. O., and Waterman, H. C., J. Biol. Chem., 44, 303 (1920). 7. Jones, D. B., Finks, A. J., and Gersdorff, C. E. F., J. Biol. Chew, 61,

103 (1922). 8. Jones, D. B., Gersdorff, C. E. F., Johns, C. O., and Finks, A. J., J.

Biol. Chem., 63, 231 (1922). 9. Jackson, R. W., and Block, R. J., J. Biol. Chem., 98,465 (1932).

10. Rose, W. C., Kemmerer, K. S., Womack, M., Merte, E. T., Gunther, J. K., McCoy, R. H., and Meyer, C. E., Proc. Am. Sot. Biol. Chem., 8, lxxxv (1936) (J. BioZ. Chem., 114 (1936)).

11. Hogan, A. G., J. BioZ. Chem., 29,485 (1917).

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Sammie PhillipsD. Breese Jones, Charles E. F. Gersdorff andTHE MAYAS, PHASEOLUS VULGARISPROTEINS OF THE BLACK BEAN OF

1938, 122:745-755.J. Biol. Chem. 

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