THE PROTEIDS OF LUPIN SEEDS.

BY THOMAS B. OSBORNE AND GEORGE F. CAMPBELL.

The lupin is a leguminous plant little known in this country except as a garden ornament. The yellow lupin (Lupinus luteus) and the blue lupin (Lupinus angustifolius), both native to Mediterranean regions, have long been cultivated in Europe because of their ability to grow luxuriantly on sandy or gravelly soils, and by their help large areas of poor, "worn out" land have been reclaimed and made agriculturally profitable, as these plants furnish abundant fodder and by the decay of their deeply penetrating roots, and especially when plowed under green, they rapidly impregnate the soil with humus and render it productive for other crops. Ritthausen (Jour. f. Prakt. Chem., 103, 78; the same, New Series, 24, 222, and 26, 422, and Die Eiweisskörper, etc., Bonn, 1872), first studied and described under the name conglutin the characteristic proteid of the lupin seed. He found that the yellow and blue lupin both yielded conglutin scarcely distinguishable in properties and only differing in composition as respects sulphur, of which his preparations from the yellow lupin contained 1 per cent. and those from the blue lupin but 0.5 per cent. Ritthausen also analyzed preparations which, from their composition, he concluded to be legumin. He stated that both legumin and conglutin are extracted from lupin seeds by salt solutions as well as by dilute alkali, and that the two proteids can be separated from each other by dissolving in alkali, then precipitating with an acid and finally treating with salt solution. On neutralizing these alkali solutions conglutin retains while legumin loses its solubility

in saline solutions.

By extracting with five per cent. sodium chloride brine Ritthausen obtained two preparations from yellow lupin having the composition given under 1 and 2; by dissolving 2 in potash water and reprecipitating, 3 was prepared; the blue lupin by extracting with salt solution yielded 4.

The proteid rendered insoluble in salt solution by previous precipitation from an alkaline liquid, by acetic acid, he found to have the following composition, these figures being the average of five quite closely agreeing analyses:

Palladin (Zeit. f. Biol., 31, 191) has recently described the properties of "vitellin" contained in seeds of the yellow lupin. In many details the results of his work do not agree with ours, but as he admits that the "vitellin" which he examined was always somewhat impure, it is unnecessary to review his statements here.

YELLOW LUPIN.

We find that seeds of the yellow lupin contain a small quantity of proteid that is soluble in pure water, a large quantity soluble in salt solutions, a small amount soluble in potash-water, and a little nitrogenous matter, presumably proteid, which cannot be extracted by these solvents.

To determine the proportions of these proteids, very finely ground yellow lupin meal was first completely extracted with warm alcohol of .868 sp. gr. in order to remove as far as possible the amides and alkaloids which occur in considerable quantities in this seed. The air dry meal before exhaustion with alcohol contained 8.16 per cent. of nitrogen. Alcohol removed 11.0 per cent. of the meal and gave a residue containing 8.49 per cent. of nitrogen, showing that 0.60 per cent. of nitrogen calculated on the original meal had been removed. The original meal accordingly contained 7.56 per cent. of nitrogen in the form of compounds insoluble in alcohol. As the seed reacted strongly acid towards litmus it is possible that a considerable quantity of alkaloids was not removed by the alcohol, but it was not desirable to add any alkali in this extraction for fear of affecting the proteids.

One thousand grams of the alcohol-exhausted and thoroughly airdried meal were treated with successive quantities of ten per cent.

sodium chloride solution until saturation of the filtered extract with ammonium sulphate showed that no more proteid was removed. The united extracts were filtered clear, saturated with ammonium sulphate, the precipitated proteids were dissolved in brine, the solution was filtered and dialyzed for several days. The abundant precipitate thus produced was filtered out, washed by decantation with water and with alcohol and dried over sulphuric acid. The substance thus obtained weighed 279.0 grams.

The solution filtered from this globulin was saturated with ammonium sulphate, the precipitate produced was dissolved in water, the solution filtered and dialyzed until no more globulin separated. By this second dialysis 15.2 grams of globulin were secured. The solution filtered from this second portion of globulin was concentrated by dialysing in alcohol and the proteid completely thrown down by adding absolute alcohol. The precipitate which resulted was then dehydrated with absolute alcohol and dried over sulphuric acid. Only 4.20 grams of substance were thus obtained, indicating the presence of very little proteid soluble in water.

The residue of meal was washed with water, which took up no proteid, and exhausted by successive applications of 2-10 per cent. potash water. The alkaline extracts were filtered clear and treated with dilute acetic acid. No precipitate resulted until a decided excess of acid had been added. The proteid thus separated was washed with water and with alcohol, dried over sulphuric acid and weighed 60.53 grams.

The meal residue was next washed with water and with alcohol, dehydrated with absolute alcohol and thoroughly air dried. It weighed 192.0 grams, showing the lupin seed to contain over 80 per cent. of substance soluble in salt solution and very dilute alkali, an amount not approached by any other seed which we have examined.

The nitrogen was determined in the various products and the results are summarized in the following table:

AMOUNT OF PROTEID EXTRACTED FROM YELLOW LUPIN BY VARIOUS SOLVENTS.

Extracted residue contained 1.51 per cent. N. or.....
Proteid extracted by alkali,

2.90 grams.

60.53 grams, contained 16 43
per cent. N. or.
279.0 grams, contained 17.86

We thus have 77.88 per cent. of the nitrogen accounted for. We will next consider in order :

I. Proteids soluble in pure water.

II. Proteids insoluble in water, but soluble in sodium chloride solution.

III. Proteids insoluble in water and in salt solution, but soluble in dilute potash-water.

I. PROTEIDS SOLUBLE IN PURE WATER.

As just indicated, water-soluble proteids occur in yellow lupin seeds in very small amount. Much the largest quantity obtained in any of the numerous extractions made was that already described, which formed 0.42 per cent. of the alcohol-extracted meal or 0.37 per cent. of the original meal. On treating this substance with water, a very considerable part was found to be coagulated. Since proteoses are not supposed to be rendered insoluble by prolonged treatment with alcohol, this insoluble matter was prob ably coagulated albumin or globulin, or possibly a mixture of both. The aqueous solution filtered from this insoluble proteid yielded a very small flocculent coagulum at 59°. The solution filtered after heating to 65°, became turbid at 67°, and flocks in minute amount appeared at 69°, which steadily increased until at 85° a very considerable coagulum had formed.

In another extraction by means of water, prolonged dialysis caused the separation of a small quantity of globulin which, when dissolved in salt solution, yielded a flocculent coagulum at 59° and but traces above 65°.