PHOSPHORUS IN VEGETABLE PROTEINS.

Transcription

1 PHOSPHORUS IN VEGETABLE PROTEINS. I. The Mode of Association of Phosphorus with the Globulins of Lathyrrus sativus (Khesari) and Vicia sativa Var. Arigustifolia (Akta). By V. V. SREMNIVASA RAU, M.Sc., A.I.I.Sc., Department of Biochemistry, Indian Institute of Seien.ce, Bangalore. Received June 30, (Communicated by Prof. V. Subrahmanyan.) Tram discovery of the biological value of organic compounds containing phosphorus has, in recent years, awakened fresh interest in the structure of natural conjugated phosphoric acids. The latter come under the group of phospho-proteins, the best known among which are casein and vitellin. It is now generally admitted that the phosphoric' acid group in these proteins is definitely in organic combination, though the precise mode of its association with the molecule has been the subject of some controversy. (Posternak, 1927; Rimington,. 1928; Posterziak. and Posternak, 1928; I,ipmann and Levene, 1932.) Of the different phospho-proteins, casein is the one that has been most studied, but its phosphorus content has, until recently, been a subject of difference among the various workers. Bosworth and Van Slyke (1914) obtained a valve of 0.71 percent.; Van Slyke and Baker (1918), 0.8 per cent. ; Carpenter (1931), 0.86 percent.; and Berggren (1932), 0.81 percent. An average value of 0.8 per cent. is now generally accepted. In the course of an investigation on the proteins of Lathyrus sativus and Vicia sativa, it was observed that preparations of globulin obtained by different methods showed varying, phosphorus contents though the distribution of nitrogen was about the same in all the cases. In view of this peculiar observation, the present investigation was undertaken with the object of determining whether the phosphorus occurs as an impurity or in combination as an integral part of the protein molecule. Lathyrus sativus (Khesari) is a leguminous annual raised in many parts of Central and Northern India. This pulse forms the chief source ofprotein for a large section of the population and, in times of famine, forms the main source of food for months together. It has been observed that a kind of paralysis, known as Lathyrism, is prevalent in areas where the pulse is consumed, the incidence of disease increasing with the proportion of Lathyrus sativus taken as food. The origin of the disease has been the subject of a number of enquiries (Buchanan, 1904; Stockman, 1917 ; Howard, 73

2 74 Y. V. Sreenivasa Rau Anderson and Simonsen, ) and general evidence would appear to point to the seeds of a leguminous weed, Viciasativa (Akta) occurring in association with Lathyrus sativus and which contains a cyanogenetic glucoside (Ritthausen, 1870; Bertrand and- Weisweiller, 1908, 1910; Mirande, 1921; Steyn, 1933) being responsible for it. The evidence is stillinconclu.sive and since the proteins form the most important food constituents in those seeds, it was considered that a study of the distribution of nitrogen may throw fresh light on the problem. Experimental. The seeds were sun dried, the husks removed and powdered and extracted with ether. In each case, 1 kg of the flour was extracted with 6 ` per cent. sodium chloride and filtered in fluted filter papers under gravity. The clear extract was dialysed in parchment paper against running distilled' water. The precipitated protein was centrifuged, washed with water and then with graded strengths of alcohol and finally with ether. The albumin, in the case of Lathyrus sativus was obtained by acidifying the d aiysate with glacial acetic acid. The globulin from Vicia sativa was obtained by dialysis' and dilution respectively. The yields were as follows : globulin from Lathyrus sativus, 25 g., albumin, 2 g.; globulin from Vicia sativa, by dialysis, 20 g., by dilution, 15 g. The analysis of the proteins was carried out according to methods outlined in a previous paper (Sreenivasa Rau and Sreenivasaya, 1933).. The results are given in the following tables :-- TABLE I. Proteins from Lath lrus sativus Globulin from Vicia sativa by Globulin Albumin f Dilution Dialysis Percentages Total Nitrogen Ash r Phosphoius 2 (as P) 0.10, Sulphur' (as. S) Micro method of Pregl. 2 Do, of Carius,

3 Phosthoyus in. i/eggetable Proteins 75 TABLr, -II. Results expressed as percentages of Total Nitrogen. Proteins from Lathyrus sativ'us Globulin from;' Vicia sativa by Globulin Albumin Dilution Dialysis Sol. Humin Insol. Huhn Amide N , Total basic N Arginine Histidine X Cystine , Lysine $ Non-basic N TABLE III. Results expressed as Percentages on protein. Proteins from Lathyrus sativus Globulins from Vicia sativa by Globulin A.Ibumin Dilution Dialysis Arginine I istidine Cystine Lysine Tyrosine, R 2.82 Tryptophane The different proteins contain about the same percentage of basic nitrogen. The globulin from Lathyrus sativus contains more of histidine and less of arginine than the corresponding protein from Vicia sativa. The tyrosine and tryptophane contents do not show any considerable variations. The albumin from Lathyrus sativus is richer in cystine than the other preparations. The biological values of the proteins are under investigation.

4 76 "Y. V. Sreenivasa, Rau The mode of association of Phosphorus with the globulins of Akta seed. The ether extracted flour (500 g.) was treated with 2 litres of 6 per cent. NaCl and shaken for half an hour. To the clear extract ammonium sulphate was added. When the concentration of ammonium sulphate was 27 per cent., a fraction was precipitated. This was centrifuged, washed with ammonium sulphate of the same concentration, dissolved in a small quantity of 2 per cent, sodium chloride and precipitated again with Am2SO4. The reprecipitated protein was dissolved in sodium chloride solution and dialysed until it was free from salt and ammonium sulphate. (Fraction I: yield, 8 g.) The filtrate from the above experiment was further treated with Am2SO 4. A second fraction separated when the concentration of Am2SO4 was 40 per cent. This fraction was similarly purified. (Fraction II: yield, 4 g.) The filtrate after removing fraction II was acidified with glacial acetic acid when a heavy precipitate came down. The precipitate was separated by centrifugation and purified in the usual manner. (Fraction III : yield, 5 g.) The phosphorus' and nitrogen contents of the three fractions were determined (Table IV). TABLE IV. Results expressed as Percentages of protein (ash and moisture free). Fraction I Phosphorus Nitrogen I Nil Ii III It was observed that fraction III which was the only one containing phosphorus was contaminated with albumin_ Salt precipitation was therefore further extended, increasing the concentration of Am2SO 4 to 100 per cent. The flour (2 kg.) was extracted with 3 litres of 6 per cent. NaC1. It was filtered through cloth and the residue again treated with another 2 litres of NaC1 and shaken for half an hour. The combined extracts were passed through gravity filters and the clear filtrate (about 4 litres) treated with increasing quantities of ammonium sulphate upto 100 per cent. The fractions which separated at different stages were reprecipitated, dissolved in 2 per cent. NaC1 solution, dialysed until free from salt and sulphate and then dehydrated (Table V).

5 Phosphorus in 'Vegetable Proteins ii TABLE V. Percentage of ammonium Yield in grains Colour sulphate Ash grey Slightly red do Pale yellow No precipitate do do.... The extract left from the above was dialysed until it was almost free from ammonium sulphate. A few c.c. of glacial acetic acid were then added and the albumin coagulated by just warming on.the water bath. The supernatant liquid was syphoned off and the residue centrifuged.. The albumin was dissolved in 2 per cent. NaCl and dialysed until free from chloride and sulphate. It was then precipitated from the dialysate by treatment with glacial acetic acid. The precipitated albumin was centrifuged and washed with water acidulated with acetic acid. It was dehydrated with graded strengths of alcohol and finally with ether. A light, white powder was obtained. (Fraction V: yield, I g.) Phosphorus and nitrogen were determined in the five fractions four globulins and one albumin. (Table VI.) TABLE VI. Results expressed as percentages on ash and moisture free protein. Fraction Phosphorus I Total Nitrogen I Globulin.. Nil 16'.60 II III IV V Albumin * * Expressed on moisture-free protein. bat not corrected foh ash.

6 Y. V Sreenivasa Rau It is clear from the above that.none of the globulins contains any phosphorus. The phosphorus content of the preparations obtained by dialysis, dilution (cold) or hot extraction and dilution should therefore be attributed to contamination of the globulins with either albumin- or phosphorus-containing compounds from the extract by adsorption. Thus, the relatively high percentage of phosphorus (0.51) in the preparation obtained by dialysis may be traced to greater adsorption of these from the extract with which the precipitated protein is in contact for 8-10 days. These observations would suggest that the so-called " phospho-globulins " from vegetable sources are largely cases of contamination with phosphorus from other sources. It is difficult to explain, at this stage, the abnormally high percentage of phosphorus and the low one of nitrogen obtained for the albumin fraction. A rough computation would indeed show that a large part of phosphorus originally associated with the globulin prepared by dialysis is now,concentrated in the small quantity of the albumin fraction. Furthermore, the nitrogen content of the preparation would suggest that it is not a true protein as is. usually understood, but is either a degradation product or, more probably,. a mixture of protein with other organic and inorganic substances including a high proportion of phosphates. Further study on the.mode of association of phosphorus with the albumin fraction is in progress gnd will form the subj ect_ of a later communication. Summary. I. The albumin and the globulin in Lathyrus sa izjus and the globulin in Vicia. sativa were prepared by different methods and:; their properties studied. 2. The globulin in Lathyrus sativus contains more of histidine and less of arginine than the corresponding protein from Vicia sativa. The tyrosine and tryptophane contents are about the same in all the cases. The albumin from Lathyrus sativus is richer in cystine than the other preparations. 3. Fractionation of the globulins of Vicia sativa by increasing salt saturation showed that none of the preparations contained any phosphorus. Small quantities of that element present in preparations :obtained by the usual -standard methods of dilation- or dialysis, especially the latter, are traceable to impurities, adsorbed or otherwise retained ; by the protein. These and the related' observations would suggest. that the whole question of phospho-globulins " of vegetable origin require careful re-enquiry. 4. After removal of the globulins (as in 3), a small q uantity of a fraction (albumin?) containing 6.7 per cent. of nitrogen and 10 per cent. of phosphorus was obtained. The, mode of association of phosphorus with this preparation is under investigation.

7 Fhos j5hovus in Vegetable Pro/ems In conclusion, the author wishes to express his thanks to Professor V. Subrahmanyan for many useful suggestions and keen interest evinced in the course of the investigation. REFERENCES Berggren, R. E. L J. Bio. Chem., 1932, 95, 461. Bertrand and Weisweiller.... Compt. Rend., 1.908, 147, 252. Ibid., 1908, 150, 180. Ibid., 1908, 151, 325. Bosworth, A. W., and Van Slyke, L. L... J. Biol. Chem., 1.914, 19, 67. Buchanan Report on Lathyrism in the Central Carpenter, P. C... Howard, Anderson and Simonsen Lipmann and Levene Mirax de Posternak.... Posternak, S.; and Posternak, T. Sreenivasa Ran and Sreenivasayaa Rimington, C..... Ritthausen.. Steyn, P. (X. Stockman.. Van Slyke, L. L., and Baker, J. C. Provinces, J. Amer. Chem. Soe,, 1931, 53, J. Indian Med. Rcs., , 12, 613. J. Bid. Chem., 1.932, 98, 109. Compt. Rend., 1921, 172, 1142, Compt. Pend., 1928, 186, Compt. Rend., 11327, 184, 909, J. Indian Inst. Sci., A, Pt. IX, Bi.odhem. J., 1927, 21 (2), Pralct. Chem., 1870,, 2, 336. " Onderstepoort. " J. Vet. Sci., 1933, 1, Ibid., Edin. Med. Jour., 1917, 19, 277. J. Biol. Chem., 1918, 35, 127.