l,j.j2
ERWIN BRAND, FRANCIS J. RYANA N D EUGENE M. DISKANT
It is cbncluded that the 4 sub-units cannot all be the same, since the minimum molecular
Vol. 67
weight is identical with the molecular weight. XEWYORK,N.Y.
RECEIVED MAY11, 1945
___..___I____
[CONTRIBUTION FROM THE DEPARTMENTS OF
BIOCHEMISTRY AND
ZOOLOGY, COLUMBIA UNIVERSITY]
Leucine Content of Proteins and Foodstuffs’J BY
ERWIN BRAND, FRANCIS J. RYANAND EUGENE M. DISKANT
I n a recent publication3 we have described a simple microbiological method for the accurate determination of leucine with the aid of the “leucineless” strain of Neurospora crassa developed by Regnery4 in Beadle’s laboratory. The reliability of the microbiological method was clearly established by a comparison with the methods involving solubility product and isotope dilution. The proportions of leucine found in crystalline egg albumin, gelatin and crystalline horse hemoglobin were practically the same by these fundamentally different methods, identical preparations having been analyzed in each case. For @-lactoglobulinwe reported3 a leucine content of 15.4%. Since then another sample of P-lactoglobulin (obtained froni Dr. C:. Haugaard) has yielded in the hands of Dr. G. L. Foster5 15.77& by the isotope dilution method. This increases our confidence in the results by the Neurospora method. We have previously3 pointed out that the bioassay for leucine with Lactobacillus arabiU O S Z I S , as carried out by Kuiken, et aL.,6 gave results for gelatin and casein only slightly lower than those reported by u s 3 Schweigert, et al.,i have since obtained a leucine value of 9.6vo for casein by the L. umbinosu ethod in good agreement with our value of 9. Reliable values for the leucine content of proteins obtained with chemical methods are available only for silk fibroin, gelatin and egg albumin which were obtained by the solubility product methodYand for horse hemoglobin and @-lactoglobulin by the isotope dilution neth hod.^ Most of the older values for leucine in the literature are of questionable reliability. Moreover, they usually refer to the sui11 of the leucine and isoleucine, although this is not always explicitly stated nor recognized. Since the ed in this paper wds done under a contract, recommended b y the Committee on Medical Kesearcti, between the Ofice of Scientilic Kesearrh and Development and Columbia University. ( 2 ) Some oi the d a t a in this paper were presented before the Division of Biological Chemistry a t t h e 106th meeting of the American Chemical Society, Pittsburgh. Pa., Sept., 1943. 131 1’. J. Kyan and P.: Brand, J. Aiol. C h e w . , 164, 161 (1944). (1) D.C. Regnery, ibizL., 164, 1.51 (l’l&4) ( 5 ) G.L. Foster, i b i d . . in prc,s. ( 6 ) K. A Kuiken, W . €J N o r m a i l , C . h l . I . y r n a n , F . Hale and I.. Iilolte?, iDid., 151, 018 ~l:l.l::l. (7) 13. S. Schweigert J ?-I. Mcrrrtlre C‘ .\ Il!i-ehje.in nnrl F. h l ‘ < f r < , i i g i,h i , ! . 156, I83 (lq,!4.Ij ,i: 5 \iqvarc. 4n.l i\’I ! ‘itvin i h u i , 150, Jl:{ (144:ii
able to extend our knowledge of protein composition by determining the leucine content of a number of common proteins and especially of certain crystalline preparations. The data are presented in Table I. The leucine values previously reported3J are included. Except as especially noted, the values are on a moisture-, ash- and sulfate-free basis. Protein Preparations We are indebted to many workers in the field of protein chemistry for samples of their preparations, as indicated below for the individual proteins. Blood Proteins.-Most of the proteins were preparedLoby the Department of Physical Chemistry, Harvard Medical School. Human serum albumin (no. 42) and bovine serum albumin (no. 456) were crystalline preparations (cf. ref. 9) and were practically homogeneous by electrophoretic and ultracentrifuge criteria (cf. ref. 11). Horse serum albumin A (the carbohydrate containing fraction, so designated by Kekwjck)12was prepared by Dr. Hans Neurath, while horse serum albumin H (the carbohydrate free fraction, (cf. ref. 12)) was prepared by Dr. Manfred Mayer of the Department of Medicine, College of Physicians and Surgeons, according to the method of Adair and Robinson. l 3 Human serum y-globulin (no. 36, 11-1, cf. refs. I), 11) was electrophoretically uniform, but was not homogeneous with respect to size (cf. ref. 9, Table 11, footnote 2 and ref. 14). The human @-globulin(no. 111-2, Prep. GL291, refs. 9, 11) was a complex mixture of human serum globulins about two-thirds of which had the mobility associated with @-globulin. Human fibrinogen (no. 81, KI, cf. refs. 9) Was clottable to the extent of about 87% of its protein content; the value reported is calculated on the assumption that the content of total nitrogen is the same in human fibrinogen as in human fibrin (9) t’, h a n d , 13. Kasscll and I,. J. Saidel, (lY44).
J . C l i ~ tI.x u e s ! . , 23, -137
(,lo) These proteins were prepared from blood collected by the .Imerican Red Cross, under a contract recommended b y t h e Committee o n Medical Research, between t h e Office of Scientific Resedrch and Development a n d Harvard University. ( 1 l j 13. J . Cohn. J . I.. Oncley, L. E. Strong, W. L. Hughes, J r . and S . H Armstrong, Jr., J . C l i n . Inwesf., 43,417 (1944). (121 I < . 4. Kekwick, Biochem. J . , 34, 552 (1938). (13) G . S Adair a n d hl. E.Robinson, ibid., 24,093 (1’330). ( 1 i) J . 1%:. Williams, h l , I,. Peterman, G. C . Colovos, M. B. Goodloe, J , I , OIITIPVnncl c: H . Armstrong. J r , , J . C l i f c . InVesf.,‘2S, 133 f l 9 4 t j
LEUCINECONTENT OF PROTEINS AND FOODSTUFFS
Sept., 1945
(16.9%). The human fibrin was prepared by Dr. H. B. Vickery from a lot of fibrinogen which had clotted spontaneously. The bovine fibrin (cf. ref. 9 Table IV) was a commercial preparation, the value given is based upon a total nitrogen content of 17.0%. The horse hemoglobin (twice recrystallized) was prepared by Dr. G. L. Foster5and the sample of human hemoglobin by Dr. R. K. Cannan. Enzymes and Hormones.-The crystalline insulin was prepared by Dr. V. du Vigneaud (cj. ref. 3). Ribonuclease, chymotrypsinogen, a,p,y-chymotrypsin, trypsinogen and trypsin were crystallized by Dr. M. Kunitz. Pepsin D was a crystalline sample prepared by Dr. R. M. Herriott and subsequently denatured. For other analytical data on these preparations see refs. 15 and 16. Animal Proteins.-Gelatin 0 was prepared by the late Dr. T. B. Osborne and obtained from Dr. H. B. Vickery. Gelatin Bg and egg albumin Bg were obtained from the late Dr. M. Bergmann and Drs. W. H. Stein and S. Moore (cf. refs. 3 and 17). Egg albumin C, crystallized once and denatured, was prepared by Dr. H. 0. Calvery. Elastin was prepared by Drs. W. H. Stein and E. G. Miller (cf. ref. 18). Vitellin was prepared by Dr. B. H. Nicolet and Mr. L. J. Saidel. Bence Jones protein was prepared by one of the authors (E. B.), Vegetable Proteins.-With the exception of the peanut proteins these proteins were prepared a t the Connecticut Agricultural Experiment Station, New Haven, and were obtained from Dr. H. B. Vickery. The sample of edestin was prepared in 1928. The sample of glycinin (precipitated by dialysis) was prepared by the late Dr. T. B. Osborne in 1920. The Zein was prepared in 1923; the Gliadin in 1939. The cotton seed globulin no. 16 was prepared in 1916, and cotton seed globulin no. 27 in 1927 by Mr. L. S. Nolan. Conarachin was prepared by The Arachin Johns and Jones from peanuts with hulls, the total peanut globulin (Johns, prep. No. 201-1917) from Virginia peanuts. These peanut protein preparations were gifts to the late Dr. T. B. Osborne. Foodstuffs.-Skim milk powder, dried brewers’ yeast and wheat flour were commercial materials. RibonucleaselYis the only protein so far investigated that does not contain I ( +)-leucine. We have controlled this observation by adding small amounts of Z-leucine to the hydrolyzates, whereupon prompt mold growth resulted. The negative results of the leucine assay in this case are therefore not due to the presence of special inhibitors in hydrolyzates of ribonuclease. Regnery4 has shown that the “leucineless” mutant
+
(15) (16) (17) (18) (19)
E . Brand a n d B. Kassell, J. Got. Physiol., 26, 167 (1941). E. Brand a n d B . Kassell, J . Biol. Chem., 146, 359 (1942). S.Moore a n d W. H. Stein, ibid., 160, 113 (1943). W.H. Stein a n d E . G. Miller, ibid., 136, 599 (1938). M. Kunitz, . I . Gen. Phg.rin/., 24, 15 (1940).
1533
of Neurospora cannot utilize the unnatural form of leucine for growth unless small amounts of natural leucine are also present (cf. ref. 3). The above experiments, therefore, indicate only the absence of 1-leucine in ribonuclease; the absence of d-leucine remains to be established. TABLE I LEUCINE COSTENT O F
PROTEINS’ Residues per mole, protein Protein, Protein, Assumed g . per moles per Kesi- molecular 100 g. 105 g. dues weight
Proteins
Blood proteins 11.9 00.7 13,7 104.4 13.0 99.1 10.1 77.0 8.9 67.9 9.3 70.9 7.1 54.2 7.1 54.2 7.5 07.2 14.7 112.1 15.7 119.7
H u m a n serum albumin, no. 42 Bovine serum albumin, no. 456 Horse serum albumin A Horse serum albumin B H u m a n p-globulin, no. GL291 H u m a n y-globulin, no. 36 H u m a n fibrinogen,no. 81 R I H u m a n fibrin Bovine fibrin H u m a n hemoglobin Horse hemoglobin
Enzymes a n d hormones Ribonuclease 0 0 Insulin 13.4 101.4 10.4 79.3 Chymotrypsinogen, no. I1 a-Chymotrypsin, no. I1 9.1 69.4 &Chymotrypsin 9.1 69.4 8.5 64.8 y-Chymotrypsin, no. I1 Trypsinogen 7.6 57.9 Trypsin 7.8 59.5 10.4 79.3 Pepsin, no. D
G4 73 54
70,000 70,000 70,000 70,000
122
171,000
75 80
66,700 66,700
0 45 29
44.60Ob 36,700
27
34,400’
32 32 50
43,000 43,000 42,020
on
Animal proteins Silk fibroin“ Gelatin 0 Gelatin Bg Elastin Egg albumin Bg Egg albumin C &Lactoglobulin (cf. ref. 21) Casein (vit. free labco) Bence-Jones protein’ Vitellin’
0.8 3.6 3.6 8.6 0.6 9.9 15.6 9.8 7.6 11.3
6.1 27.4 27.4 65.6 73.2 75.5 118.9 74.7 57.9 86.2
Vegetable proteins Edestin Gliadind Zein“ GI ycininc Arachin conarachin’ T o t a l peanut globuline Cotton seed globulin no. 16# Cotton seed globulin no. 27‘
+
7.4 6.5 15.4 8.4 8.0 s.1 6.8 6.4
56.4 49.6 117.4 til.0 61.CI
ti1.s 01. Y 48 8
Foodstuffs’ Skim milk powder Dried brewers yeast Wheat flour
3.5 2.9 0.8
All values are on a moisture-, ash- and sulfate-free basis, except as otherwise indicated. * From unpublished analytical data of E. Brand and L. J. Saidel (molecular weight by ultracentrifuge 46,000 (cf. ref. 20). From unpublished analytical data of E. Brand and €3. Kassell. Taken from Moore and Steins and determined by the solubility product method. Not corrected for ash. 1 Not corrected for moisture and ash. (20) G . L. Miller a n d K. J. I. Andersson, J . B d . Chcm., 144, 459 (1942). (21) E. Brand, L. J. Saidel, W. H. Goldwater, R . Ka-ell and F. J. Kyan, THISJOURNAL, 67, 1.531 (1915)
ROGERAD~OIS,E;. H. CHENAND S. LOEWE
1534
Vol. 67
Of all the proteins so far investigated, silk pared to 15.4% by the Neurospora method. fibroin has the lowest leucine content (O.Sso, Ribonuclease is the only protein in which no determined by the solubility product metlicld*), leucine could be detected. gelatin being next with 3 . V r . Of all the proteins so far investigated, silk fibroin lias the lowest leucine content (0.8%), Summary gelatin beiiig next with only 3.6y0. Leucine accounts for a n appreciable part of the The leucineless strain of iVeurospora crassa was used for the determination of leucine in hydroly- molecule in zein (15.4%), bovine (13.7%), husates of a number of proteins and of a few food- iiim (11.9%) and horse (13.0%) serum albumin, horse (15.7'73 and human (14.7y0)hemoglobin, stuffs by the technique described previously. Additional evidence for the reliability of the irisulin (13.4($/0)and 6-lactoglobulin (15.6%). Chymotrypsinogen, insulin and &lactoglobulin microbiological method has become available in the case of @-lactoglobulin, for which a leucine coiitain 29, 45 and 50 residues of lrucine, recontent of 15.75; has been found by Dr. G. L. spectively, per mole of protein. Foster by the isotope dilution method as com- Nlan YORK,N. Y. RECEIVEDMAY28, 1945
[CONTRIBUTION FROM
THE
NOYESCHEMICAL LABORATORY, UNIVERSITY OF ILLINOIS, AND THE PHARMACOLOGY DEPARTMENT, CORNELL UNIVERSITY MEDICAL COLLEGE]
Tetrahydrocannabinol Homologs with a s-Alkyl Group in the 3-Position. XVI' BY ROGERADAMS,K. H. CHENAND S. LOEWE Modification of the synthetic tetrahydrocannabinol molecule (I) by changing the substituents in the 6- and 9-positions invariably produced compounds of lower marihuana potency. In the 3position, however, an increase or decrease in the physiological activity could be attained with variation of the size of the alkyl group. Activity increased progressively from the methyl to the n-hexyl and then decreased in the higher homologs. 1 . 2 . 3 CHa
OH
I
C'H? CH, rI
With two exceptions the molecules previously studied had nornial groups in the &position. Todd3 described the isoamyl and isohexyl honiologs and observed that they had only negligible activity by the Gayer test. The investigation has now been extended to a series of molecules with alkyl groups containing a secondary carbon attached to the benzene ring (11). In these substances the physiological ac(1) For previous paper, see Adams, Loewe, Theobald and Smith, THISJOURNAL, 64, 2663 (1942). (2) Adams. Loewe, Jclinek and Wolff, ibid.. 63, 1071 (1941). (3) Russell, ?'mid, n!iikinson. Macdon,rld .ind Wonlfe, J. Chcm. Scv , 82C ( l Q 4 l )
tivity showed a marked increment in value over the corresponding normal alkyl derivatives. In Table I a comparison is given; all products were tested by the procedure previously described. PHARMACOLOGICAL
TABLE I ACTIVITYOF TETRAHYDROCANNABINOL HOMOLOGS
3-Substituent
No. of expts.
1 -GHp-n 4 2 -c6HI1-n 20 3 -CH(CHa)CsH7-n 11 4 -CH (C2H5)CaH7-n 11 7 --CH(CHa)C4Ho-n 7 ti -CH(n-CaH7) C4Ho-n 8 7 -C&-n 7 8 -CH (CH,) C6Hi1-n 13 9 -C7H16-n 10 10 -CH (CH ) CsH18-n 10 1 1 -CsH17-~ 7 12 T e t r a h y d r o c a ~ i n a b i n o l ~ from cannabidiol (from 20 American hemp) 13 Katural tetrahydrocannabinols acetate (from 5 charas) 14 Natural tetrahydrocannabinols by hydrolysis of 13 15 15 I'ulcgone 5-(l-methylbutyl)-resorcinol 5 16 Pulegone 5-(n-amyl)resorcinol 11
+ +
Potency
0.37 * 0.12 1.00 standard 1.84 * 0.13 1.67 * 0.33 3.66 * 0.33 3.17 * 0.25 1.82 * 0.18 4.85 * 0.82 1.05 * 0.15 16.4 * 3.67 0.66 * 0.12
7.3
*
0.89
14.6
*
1.05
7.8
*
0.47
0.45
*
0.05
0.58
*
0.12
Examination of Table I reveals several interesting facts. In all cases, the secondary groups (4) Adarns, Tnewe, Smith and McPhee, THISJOURNAL, 64, 694 (19421. (.7) WVnlln?r, I r a t C I i ? t t . Levine and Imewc, ibid.. 64, 26 (1942).
