2644
COXMUNIC ATIONS
TO THE
EDITOR
Vol. i 8
120 m m . j were added to the ethanol. In the course of 15 tilled very slowly for 2 hr. through a 1U-in. helices packed minutes the temperature rose to 40' and after 1 hr. two drops column; 10 ml. of distillate was collected. The mixture of concentrated hydrochloric acid were added to neutralize was cooled and acidified with 25.5 ml. of Concentrated hythe caustic. The ethanol was distilled off a t water pump drochloric acid and then made strongly alkaline with sodium vacuum on the steam-bath, and the residue was distilled hydroxide (21 g., 0.6 mole) dissolved in water (50 ml.). giving 17 g. (94% yield) of material boiling a t 113-118" This solution was extracted with two 100-ml. portions of (0.05 mm.). This distillate crystallized almost inimedibenzene, and the combined benzene extracts were dried over ately and melted a t 57-59'; recrystallization from petroanh>-drous potassium carbonate. The benzene was evapleum ether ( b . p . 60-70") did not raise this melting point. orated a t water pump vacuum on the steam-bath and the A n a l . Calcd. for CgH12?;?O2: C, 60.43; H, 6.59; S, residue was distilled, giving 15 g. (82.57, yield) of material boiling a t 95" (0.1 mm.) which crystallized and melted a t 15.55, Found: C, 60.15; H, 6.7%; S , 15.35. 73-76 O. A small sample of this material was converted to the A n a l . Cdlcd. for CYHldSlOL: C, 59.38; H , 7.74; S , semicarbazone in the usual manner. After crystallization 15.38. Found: C,59.16; H , 7.93; S,15.26. from water the semicarbazone melted a t 179-180". 3-(3-Methyl-6-pyridazonyl-l)-2-methylbutanol-2 .--MethAnal. Calcd. for CloHI:,SiO?: S, 29.55. Found: S, ylmagnesium iodide was prepared in the usual manner from 29.0. magnesium turnings (2.64 g., 0.11 atrxii) and methyl iodide Alkylation of 3-Methyl-6-pyridazone with 3-Chloro-2-bu(17.1 g., 0.12 mole) in absolute ether (125 ml.). This tanone.-To a solution of sodium (4.2 g., 0.18 atom) in solution was cooled to 10" and with good stirring there W;LS ethanol (200 ml.) there was added 3-methyl-6-pyridazone added slowly a solution of 3-(3-mcthyl-O-pyridazonyl- 1)(18.0 g., 0.16 mole); this mixture was cooled to below IO", butanone-2 (16 g., 0.089 mole) in absolute ether (50 m l . ) ; and 3-chloro-2-butanone (21.3 g., 0.18 mole) was added a solid complex was formed which was decomposed by the dropwise with good stirring. The mixture was heated a t addition of ammonium chloride ( 7 g.) in water (20 ml.). reflux for 2 hr. and then evaporated at water pump vacuum An excess of anhydrous potassium carbonate was added to on the steam-bath. The residue was taken up in benzene the mixture and the ether was separated and the solid w a s (IO0 ml.), and this solution was washed with two 50-1111. washed with two 100-mi. portions of benzene. The henzeric portions of 30Yc potassium carbonate solution. The benand ether solutions were combined and evaporated a t water zene solution was dried over anhydrous potassium carbonate pump vacuum on the steam-bath. The residue was disand then evaporated under vacuum. The residue was dis- tilled giving 12 g. of material boiling at 80-90" (i1.W mm.). tilled, giving 8.7 g. (30% yield) of material boiling a t 90This distillate was heated under reflux for 2 hr. with a solu100' (0.1 mm.), n 2 6 ~1.5063. n Z 5 D of 3-(3-metliy1-6-p~-- tion of semicarbazide hydrochloride (6.9 g.) and anhydrous ridazonyl-l)-butanone-Z is 1.5226. Seeding this material sodium acetate (4.9 9.) in water (30 ml.) in order to separate with 4-(3-meth~-l-6-pyridazon).I-l)-butanone-2 did not inurireacted ketone as the semicarbazone. The rewlting duce crystallization. solution was evaporated to dryness under water pump A sample of this material was converted to a semicarbavacuum on the steam-bath and the residue was treated with zone in the usual manner. The crude semicarbazone melted benzene (100 ml.), and this suspension was filtered. Evapa t 179-183'; after three recrystallizations from water it oration of the benzene solution followed by distillation gave melted a t 191-197", no depression when mixed with the i g. (4ly0yield) of material boiling a t 95-100D (0.02 mm.) semicarbazone of 3-(3-methyl-6-pyridazm~-l-l)-but:inone-2. which crJ-stdllized and melted a t 53-55', A small sxmple A mixed melting point with the semicarbazone of 4-(:3-methafter recr!-stnllizutii)ti from petroleum ether (b.p. C,0-70°) yl-6-pyridazonyl-l)-butanone-2 melted a t 160-175'. melted ;it t57.5-5X.0°. Preparation of Alcohols. 3-(3-MethyI-6-pyridazonyl-l j.lnci/. Calcd. for CIIIHl8S?O2: C, 61,211; H , 8.22; S, butanol-2.-A mixture of dry isopropyl alcohol (100 ml.), 14.28. Found: C , (il.:31; H , 8.08; S,14.2[1. aluminum isopropoxide (freshly distilled) (20.1 g., 0.1 mole) SEK. YORK11, SEIV IT^^^^ and 3-(3-methyl-R-pyridazoiiyl-l)-butanone-2 was di+
COMMUNICATIONS THE MECHANISM OF PEPSIN DENATURATIJK
'ro
THE EDITOR
for both the iiatil e and denatured forms of pepsin. I n 0.10 I'/2 phosphate buffer the S,",, was 3.08 a t In 1930 Northrop' observed t h a t the inactivation PH G.0 and 2.03 a t pH '7.0 for the two forms, reof pepsin was paralleled by the formation of acid spectively. To distinguish between frictional and mass insoluble pepsin. Subsequently Philpot3 reported that the sedimentation constant (S) of pepsin changes in pepsin during inactivation, light scattering and viscosity methods were employed. At showed a gradual decline between pH 5 and 11. The gradual decline in sedimentation constant PH 6.42 the reduced intensity (R = 190r2/I~)dethat Philpot observed has now been shown to rep- creased uniformly to about 60y0of its initial value. resent the change from native to denatured pep- \Vhen the log of the fractional residual scatter {logsin and occurs over a very narrow range of pH, con- (Pt - R,),'!Ro - R,)] was plotted against time a forming to the enzyme kinetics of inactivation4 linear relationship was observed. The kinetics Single symmetrical boundaries, which spread a t agreed closely with that determined from the rate similar rates, were observed in the ultracentrifuge of formation of acid insoluble pepsin. The viscosity of pepsin solutions was found to (1) Aided in part b y grant No. C-1974 from t h e Piational Cancer increase on inactivation. The rate of increase in Institute of t h e National Institutes of Health, Public Health Service, and b y an Institutional Grant from t h e American Cancer Society. viscosity paralleled the rate of enzyme inactivation ; (2) J. H. S o r t h r o p . J . Gen. Ph$'siol., 13,739 (1930). the data appear in Table I. 1 3 ) J S t . I,. Philpot, Biockrn7..I , 29, 21518 ( 1 9 3 5 ) . Sirice optical rotatory changes c:~n serve :IS U I f 4 1 I . Steinhardt, K c l . I l i i n i l r 1'idr;iskoh S e i $ / iih .Iliif. t y ? dfrt!il , iiidicator of molecular structural alterations in pro14,n u . 1 1 1917;
Sir:
COMMUNICATIONS TO THE EDITOR
June 5 , 1956
2645
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 soluwhich manifestly involves the rupture of carboxyl tions, which were near pH 5 5, t o their experimental PH values 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.
