COMMUNICATIONS TO THE EDITOR
June 5 , 1956
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shape of pepsin decreases the electrostatic free energy of ionization.'O I t thus appears that the loss of pepsin enzyme activity is accompanied by the appearance of both hydrophilic and hydrophobic groups, gross changes TABLEI T H EKINETICS OF PEPSISISACTIVATIOS BY FOUR DIFFERENT in the mass and shape of its molecular kinetic unit and that the bio- and physico-chemical alterations METHODS are expressions of a single rate-determining process, A11 solutions were adjusted from unbuffered stock solutions, which were near pH 5 5, t o their experimental PH val- which manifestly involves the rupture of carboxyl ues b y the addition of relatively small volumes of concen- linked hydrogen bonds.
teins and polypeptide^,^ the rate of increase in levorotation was compared with the rate of enzyme inactivation and found t o be indistinguishable.
trated buffers The pH of the buffers were only slightly higher than the pH of the experiment T h e pepsin concentration is for t h e total weight of sample. These crystalline preparations of pepsin (U'orthington Biochemical Corporation) contain about 20y0 split products The light scattering experiments were performed a t room temperature, which was 28"; in the other procedures the temperature was controlled at 25 0' Xn Ostwald viscometer was used with a flow time of 89 2 sec The optical rotatory change was for a 2-dm. cell. Tris buffer is tris-(hydroxymethy1)-aminomethane. Method
Pepsin g. /lo0 ml.
$H
Buffer
.M
Set observedk X IO2 change (min.?)
(10) As t h e NaCl concentration is Increased t o 1 M the acid liber This would ated decreases t o about 75% of its value in 0 15 M NaCl leave about 4 moles of acid originating from hydrogen bonded groups
DEPARTMENT OF PATHOLOGY A N D OUCOLOCY USIVERSITY OF KANSAS MEDICAL SCHOOL KANSAS CITY,KANSAS H A R O L EDELHOCH D RECEIVED MARCH 30, 1956 BIOSYNTHESIS OF T H E PYRIDINE RING OF NICOTINE'
Sir:
X biosynthetic connection between nicotinic acid, or its supposed antecedents, and the pyridine ring of nicotine has been proposed.2s3,4 However, isotopic 2 15scosity 0 . 3 3 7 . 1 0 Imidazole 0.015 +3.1 sec. 4 . 8 tracer experiments using carboxyl-CI4nicotinic5and KaC1 0.009 anthranilic6 acids and tryptophan-P-CIJ have 3 Optical 0.80 7.08 Imidazole 0 . 0 4 5 -0.25" 5,i failed to yield supporting evidence. Recently, rotaKSOI 0.008 Dewey, Byerrum and Ball8 and Leetegreported that tion the pyrrolidine ring of nicotine, and in particular 4 Acid 0 . 4 2 6 . 6 4 Tris 0.023 5 . 6 H / 3.4 the carbon atom a t position 2' in the pyrrolidine liberaSaCl 0.146 mole ring, is derived from ornithine. If this is correct, tion pepsin) obviously the labeled atoms of the three compounds listed could not be retained in the newly formed Finally in the conversion of pepsin from a cata- nicotine molecule. Therefore, the experiments lytically active protein t o an inactive form (in the cited ab0ve~7~J are inapplicable. p H range 6.64-6.97), about 3.6 moles of hydrogen Ring-labeled H3-nicotinic acid was prepared by ions are liberated per mole of enzyme. In four ex- neutron irradiation of a mixture of the acid and periments in this p H interval, the first order veloc- lithium carbonate, lo with carboxyl tritium being ity constants agreed with the rate of inactivation6 removed during the purification of the acid. Nicowithin 5y0. The unmasking of these acidic groups tinic acid containing CI4 in both ring and carboxyl is reflected by appreciable differences in the titra- positions was obtained by neutron irradiation tion curves of native and denatured pepsin above of nicotinamide. l 1 p H 6. These groups are displaced t o lower pH valWe have supplied these two preparations to ues in the denatured form. sterile cultures of excised roots of Turkish tobacco5 The acid formed in the inactivation of pepsin can and have found substantial amounts of radioactivity be most readily accounted for if it comes from hy- in the nicotine produced by the roots during their drogen-bonded carboxyl groups. The intrinsic pK growth (Table I). Lesser incorporation of C'.' of carboxyl groups in a number of proteins is near label was partly due t o the use of limiting amounts 4.5.' The pK of these groups in pepsin are in- of nicotinic acid, and also to the loss of nicotinic creased from their intrinsic values both by a sizable acid carboxyl ~ a r b o n . ~ Even in this case, however, contribution from the electrostatic free energya and (1) Work performed under t h e auspices of t h e U. S. Atomic Energy by the free energy of hydrogen b ~ n d i n g . ~I t Commission a t Brookhaven National Laboratory and a t Columbia should be noted that part of the 5.6 moles of acid University under Contract No. AT(30-I 1-1778. formed probably comes from non-hydrogen bonded (2) E. Winterstein and G. Trier, "Die Alkaloide," 2nd ed., Bornionizable groups since the change in the mass and traeger, Berlin, 1931, p. 1031. 1 Light 0 . 3 5 6 . 4 2 Cacodylic 0.023 -60!& scatter KNOs 0.136
4.4
'
(5) P. Doty and J . T . Yang, THISJ O U R N A L , 78, 499 (1950); R . B. Simpson and W. Kauzmann, ibid.,76,5139 (1953). (6) M. L. Anson, J . G e n . Physiol., 2 2 , 79 (1938). T h e test was modified t o t h e extent t h a t t h e concentrations of split products were determined by their absorption a t 280 mp. ( 7 ) C. Tanford, S. A. Swanson and W. S. Shore, T H I S J O U R N A L , 77, 0414 (1955), cf. Table 111. ( 8 ) G. E. Perlmann, in "Advances in Protein Chemistry," &I. I,. Aason, K. Bailey and J. T. Edsall, Vol. X, Academic Press, Inc., New York, N. Y . , has reported t h a t t h e isoelectric point increases from a value near DH 1.0 t o 1.7 when t h e single phosphate group of pepsin is removed by enzymatic means. (91 11. Laskowski, J r . , and H . 4 Scheraga, THISJOCRNAI., 7 6 , A305 (1USdJ.
(3) G. Klein and H. Linser, Planla, 20, 470 (1933). (4) R. Dawson, Plaiil Physiol., 14, 479 (1939). ( 5 ) R . Dawson, D. Christman and R . C . Anderson, T H I SJ O U R N A L . 76, 5114 (1953). (6) R . Dawson a n d D. Christman, unpublished d a t a . (7) K. Bowden, S a l u v e , 172, 708 (1953). (8) L. Dewey, R. Byerrum and C. Ball, Biocheni e l Biophys. A d a , 18, 141 (1955). (9) E . Leete, C h e m a n d Ind., No. 19, 537 (1955). (10) R. Wolfgang, F. Rowland and C . T u r t o n , Science, 121, 715 (1955). (11) A. Wolf and R . C. Anderson, THISJOVRNAL, 77, 1009 ( l 9 5 5 ) , r f . R . C Anderson. 13. Penna-Franca and A. \Volt, Brookhaven National Laboratory Quarterly Progress Report, Octoher 1-December 31, 1054.
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COMMUNICATIONS TO THE EDITOR
Yol.
is
a quite significant amount of radioactivity appeared in the nicotine.
quence of D and L configurations' and others apparently containing sequences of adjacent mers of like c~nfiguration.~,~ TABLE I The closely related problem of preparing a polyACld Sicotine mer or copolymer with an excess of one configurasoecific Sicosuecific activity tine activity tion in the backbone of the polymer chain and ex(d.p.m./mg (d.p.m./mg. yield, C or HI mg. C or €1) Compound supplied hibiting optical activity was first attacked unsuccess fully about sixty years Since then further 1 75 mg. nicotinic acid-H? 6 4 X 1V 25 1 73 x 1 0 6 attempts have been made by initiating polymeriza2 44 mg nicotinicacid-H3 6 4 X 10fi 25 1 33 x l o b tion with optically active acyl peroxidess and by "1 102 3 9 mg. nicotinic acid-C*1910 polymerizing and copolymerizing optically active Oxidation of the tritium-labeled nicotine (sample m ~ n o m e r s . ~Ailthough ,~~ the latter method might 1, Table I) with hot concentrated nitric acid yielded be expected to induce an excess of one configuration nicotinic acid having a specific activity of 2.97 X during polymerization, removal of the optically aclo6 d.p.m./"g. H. Owing to the possibility t h a t tive centers initially present has so far left inactive isotope exchange might have occurred under such polymers. drastic conditions, the oxidation was repeated with I n the following synthesis we have found evidence aqueous potassium permanganate as reagent. of induced asymmetry during vinyl polymerization : Nicotinic acid was obtained which had a specific I-a-inethylbenzyl alcohol (1) ( ( M ) 2 5-41.3', ~ activity of 4.74 X lo6 d.p.m. 'mg. H. This can be n = - l i . O o , 1 = 0.3 cm.)ll was allowed to react compared t o the activity of the nicotine (sample 1, with inethacrylyl chloride13 in pyridine. 1-aTable I ) , which is 4.S4 X IO6 d.p.m.//mg. H as llethylbenzyl methacrylate (11) (b.p. 92' a t :3-4 nl'cotinic acid (assuming the pyrrolidine hydrogens inm.) was isolated by conventional methods in 63% to be inactive), and thus represents an almost coni- yield. (Anal. Calculated for CI2Hl4Oa:C, 75.7s; plete recovery of tritium from the nicotine. H ; 7.4. Found: C, 73.8; H , 7.7. [LI]"D -7S.S'. Further details of these and other supporting u = -20.74'; I = 0.3 dcm.). experiments will appear in a later paper. (11) was polymerized in the absence of air in I t is thus probable t h a t the pyridine ring of peroxide-free dioxane a t 33' for 32 hours or more. nicotinic acid is a biosynthetic precursor of the a ,a'-.%zobisisobutyronitrile (XBIN) photosensitized pyridine ring of nicotine and of nornicotine12 and by irradiation from a Type AH4 Hanovia Xercury perhaps also of the related tobacco alkaloids, lamp (General Electric Co.) was used as i n i t i a t ~ r . ' ~ myosmine, anabasine, anatabine, etc. I t is of The polymer (111) isolated by precipitations in peinterest to note t h a t this work, together with that troleum ether and freeze drying in dioxane was of Dewey, Byerrum and Ball8 and Leete,g consti- found to have an ultraviolet absorption spectruni tutes the first recorded biosynthetic pathway for a with peaks a t 232, 23s and 264 inp, characteristic of plant alkaloid. I t is further noteworthy t h a t a the a-methylbenzyl group, and a negative rotation. universally distributed cofactor of biological cat- ( [ 1 1 ] 2 5 ~ 147, a = -0.87, 1 = 0.3 dcni., c = alysts and an important amino acid metabolite 2.2c/c in dioxane.) Calcd. for (C12H1402),,: C, -should be involved as intermediates in the syn- i o . 7 ; H, 7.4. Found: C, 75.2; H, 7.7. theses of one of a class of substctnces for which no The a-methylbenzyl groups were removed by biochemical or physiological significance has yet heating 0.300 g. of (111) with 2 g. of phosphonium been adduced. iodide (PHJ) in acetic acid a t 60".'4 The peaks of the ultraviolet absorption spectrum (12) R . Darvson, THISJ o c n x A L , 67, a03 f l O 4 J i DEPARTMENT OF BOTANY RAYF. DAWSON of the reduced polymer disappeared into the backCOLUMBIA UXIVERSITY DAVIDR . CHRISTMAS ground absorption on reduction ; the analysis inR . CHRISTIAS .%SDERSOS SEW YORK27, S.Y . , and dicated complete removal of the ester groups (calcd. DEPARTMENTS OF CHEMISTRY ASD BIOLOGY MARIEL. SOLT for (C4H602),,:C, 33.9; H , 7.0. Found: C, t5t5.7; SATIONAL LABORATORY .%MEDEO F. D'.\DAMO BROOKHAVEN ULRICH~I*EISSH, 7.3) and as expected'j the resulting polymer (IV) V P T O S , L. I.,s.Y . showed no optical activity when observed under RECEIVED - ~ P R I L5. 1956 the same conditions as above. IYhen (11) was similarly copolymerized with maleic anhydride in a l : 3 molar ratio of ester to maleic A N ASYMMETRIC COPOLYMER SYNTHESIS (4) C . E. Schildknecht, e / n I . , I n i f . E z z . Cheti?., 40, 2104 (1948); 4 1 ,
Sir:
The multiple possibilities for stereoisomerism in vinyl polymers containing monosubstituted mers was early used by Staudinger' to explain their generally amorphous character and by Huggins? to explain the dependence of their physical properties on temperature of p ~ l y m e r i z a t i o n . ~ Practical methods have now been devised to prepare more crystalline monosubstituted vinyl polymers, which are said to contain an alternating se( 1 ) H . Staudinger, "Die Hochmolekularcn organische Vurliindun~ gen," J u l i u s Springer, Berlin, 3031), p 1 1 4 . ( 2 ) .\I 1. H u ~ g i u s~. ' I I I S J O ~ J R N A I ,6, 6 , l9!ll (I!J44). : , < I '1'. A l f r e y , A . I l i r r t o L i c i a n d H . hl'irk, i b t a i . , 6 6 , ! 2 3 ' , l ( l ! l l , l ~ .
1998 ( 1 9 4 9 ) ; 4 1 , 2381 (1949). C . N a t t a , e l ai.,THISJ O U R N A L , 77, 1708 (11133). G. h-atta, .I. ~
DenBerghe, 11'. J . Dulmage a n d 1;. I
