480
NOTES
contents of one flask were used in one kinetic determination. The concentration of I was about 0.00049 mole, that of I1 about 0.0004 mole and of I11 0.00024 mole, the largest amount that could be dissolved. The concentrations did not affect the rate constants within that range, because the use of lower concentrations for the first two compounds (0.00038for I and 0.00027 for 11) did not alter them. Rate constants were calculated from the integrated form of the first-order rate equation; the errors reported in Table I are average deviations. Data for one determination are listed in Table 11. N'ith an initial concentration of 0.0002736 of bromo compound the rate constant was 2.81 X set.-'.
TABLE I1 THE REACTIONBETWEEN 3-NITRO-4-BROMOBIPHENYL PIPERIDINE IN DIOXANE AT 25" Time, sec.
Moles X 104 of bromide
3600 7200 9000 12600 15720 18060 21660 25020 31440 31980 50100
4.042 4.020 4.017 4.017 4.009 4.042 4.060 4.070 4.048 4.035 4.042
(a
- x)
X 104
3.629 3.267 3.099 2.799 2.538 2.412 2.182 2.022 1.644 1.631 0.946
\VITH
k X 106, sec. -1
(2.99) 2.88 2.88 2.87 2.91 2.86 2.87 2.80 2.87 2.83 2.90
Results qualitatively similar to those reported in Table I were obtained in preliminary experiments conducted in boiling benzene (40 ml.) to which 4 ml. of piperidine had been added. DEPARTMENT OF CHEMISTRY BRYNMAWRCOLLEGE BRYNMAWR,PENNA.
Absence of Wall Effects in a Typical a-Chymotrypsin Catalyzed Hydrolysis'
VOl. 77
buffer, under conditions where the enzyme concentration was maintained at 0.0266 mg. proteinnitrogen/ml., i.e., cu. 0.76 X M,9 the initial specific substrate concentration a t 10 X M, and where only the nature or the surface area of the container was varied. I t will be seen from the data presented in Table I, for experiments 1 to 3, inclusive, that there is no significant difference in the initial velocities when either Pyrex or Kimble glass or polyethylene containers of equivalent surface area were employed. Furthermore, the addition of powdered Kimble glass to either the Kimble or Pyrex glass containers, or powdered Pyrex glass to the Pyrex container (cf. Table I, experiments 4 to 8, inclusive) was without effect even though the increase in surface area was of the order of twentyfold in the extreme cases. TABLE I ~~HYMOTRYPS~ CATALYZED N HYDROLYSES OF
ACETYL-I.-
TYROSINHYDROXAMIDE~
Expt.
1
2 3 4 5 6 7 8
Nature of vessel
Pyrex glass Kimble glass Polyethylene Kimble glass Kimble glass Kimble glass Pyrex glass Pyrex glass
Powd. glass added Nature Weightb
.... . . ....
Total areac
.. ..
wd
22 22 22 83 412 61 81 406
0.160 .159
. . . .. , . .156 Kimble' 0.30 ,153 Kimble 1.50 .157 Pyrex' 0.20 ,160 Pyrex 0.27 ,157 Pyrex I .33 ,157 Average of all values 0.157 a In aqueous solutions a t 2 5' and pH 7.62 and 0.3 A I in the THAM component of a THAM-HC1 buffer, [ E ] = 0.0266 mg. protein-nitrogen/ml., [SI0 = 10 X M. * In g. In units of cm.2 d In units of IO-* M/min. a 150-200 mesh, density 2.5 g . / m . s . f 150-200 mesh, density, 2.25 g./cm.s.
BY RICHARD A. BERNHARD AND CARLNIEMANX~ AUGUST20, 1954 RECEIVED
Therefore, i t may be concluded from the results of this study that, for the case a t hand, wall effects In the past3v4it has been tacitly assumed that a- are experimentally unimportant under the condichymotrypsin catalyzed hydrolyses, which are con- tions which are employed generally in in vitro studductedunder the conditions ordinarily used in in vitro ies with a-chymotrypsin and that it is reasonable studies of the mode of action of this enzyme, pro- to assume that in all a-chymotrypsin catalyzed receed entirely in solution and that wall effects, aris- actions which are studied under these conditions ing from the interaction of the reactants with the the reaction can be postulated as proceeding in walls of the container, are unimportant. However, solution in so far as can be determined within the it appears that no one has ever determined whether limits of experimental error. The average of the eight values of YO which are or not the above assumption is a valid one. Theremole/min., may fore, we have, in this investigation, examined a rep- given in Table I, i.e., 0.158 X resentative a-chymotrypsin catalyzed hydrolysis be compared with the value of 0.166 f 0.028 X rnole/min. calculated on the basis of [E] = with respect to possible wall effects arising from 0.0266 mg. protein-nitrogen/ml., [SI0 = 10 X the nature and surface area of the container. 3 X The initial velocities were determined by the iM,K s = 43 f 4 X 10-3 M a n d k3 = 33 method of Jennings and Niernann from both zero- 10-8 M/min./mg. pr~tein-nitrogen/ml.~.The fact and first-order plots,6 for the cr-chymotrypsin that these two values agree, within the limits of catalyzed hydrolysis of acetyl-L-tyrosinhydroxam- experimental error, can be taken as evidence of the ide,4*6-7 in aqueous solution a t 25' and pH 7.6 and consistency of the value of vo reported in this com0.3 M in the THAM8 component of a THAM-HC1 munication with those determined earlier.4s6*7
*
(1) Supported in part by a grant from the National Institutes of Health, Public Health Service. (2) T o whom inquiries regarding this article should be sent. (3) H. Neurath and G. W. Schwert, Chcm. Revs., 66, 69 (1950). (4) R. J . Foster and C. Niemann, THISJ O U R N A L , 7 7 , in press. (5) R . R . Jennings and C. Niemann, i b i d . , TO, 4687 (1953). (6) D S.Hogness and C. Niemann. i t i d . , 7 6 , 884 (1953). (7) R . 1. Foster a n d C . Niemnnn, Pror. N n t l . A r n d . S r i . . 39, R W (IU53). ( 8 ) Tris-ihydroxymethylj-nminometiiane
Experimental Containers.-The Pyrex and Kimble glass containers used in this study were standard IO-ml. glass-stoppered volumetric flasks which had been cleaned with hot water containing a detergent and then thoroughly washed with distilled water. The polyethylene container was a 60-ml. screw cap bottle which had been treated similarly. (9) Based upon a molecular weight of 22.000 and nf 16.0'% for monomeric a-chvmotrypsin.'
R
nitroyrn
mntrnf
NOTES
Jan. 20, 1955
481
Powdered Glass.-Pyrex and Kimble glass tubing was their respective isotopic distribution pattl 1s deterground in an iron mortar and pestle, the product which mined by degradation studies. passed a 150-mesh screen collected on a 200-mesh screen, Experimental digested with concentrated hydrochloric acid for 48 hours a t 25-30', thoroughly washed with distilled water and dried Isolation of Fractions.-A solution containing 1.1 g. of a t 145' for 2 hours. In the calculation of surface areas an g l ~ c o s e - 2 - Cwith ~ ~ a total activity of 1.66 X lo5 c.p.m. was average diameter of 0.0088 cm. was assumed. The density passed through a column 1.6 cm. diameter and 32 cm. long of Pyrex glass was taken t o be 2.25 g./crn.8 and that of the charged with 39 g. of IRA400 (hydroxyl form). The abKimble glass 2.5 g./cm.J. sence of radioactivity in the efffuent indicated that there Enzyme and Specijic Substrate.-The a-chymotrypsin was complete retention of the sugar on the column. was an Armour preparation lot No. 10705. The acetyl+ After standing 24 hours at room temperature the stoptyrosinhydroxamide, m.p. ca. 140' dec., [aIZ5D 37.0" ( c pered column was eluted with 1 N (NH4)2COa. After the 570 in water), was prepared essentially as described by removal of ( NHa)&03 by partial evaporatlon, the eluate Hogness and Niemann.6 subsequently was passed through an IR-112 cation exchange Enzyme Experiments.-All reactions were conducted as column to remove any remaining ammonium ions, and described by Hogness and Niemanns except that the im- through an IR-4B weakly basic exchange column to remove proved analytical procedure described by Foster, Jennings any acidic constituents. The effluent from the latter thus and Niemann'o was substituted for the one used earlier.@ contained only the sugars and other neutral components In every case plots of both ( [SI0 [SIt) vs. t and In [SIO/[S]~ and gave a total activity of 1.07 x 10s c.p.m. or 64% of the us. t were made and then corrected as described by Jennings original activity. and Niemann6 using a value of K s = 43 X lo-* M . The The original column was eluted with 2 N H&Oc to recover values of uo given in Table I are the averages of those ob- the acidic products retsined on the IRA-400. This was tained from the corrected zero and first order plots which in subsequently combined with the 2 N HsSO, eluate of the no case differed by more than =t0.005 X lo-' Mlmin. IR-4B column. Organic acids then were recovered from the combined eluates by evaporation and exhaustive ether ex(10) R. J. Foster, R. R. Jennings and C. Niemann, THIS JOURNAL, traction. The mixed acid fraction so obtained had an ac76, 3142 (1954). tivity of 2.48 X 104 c.p.m. or 15% of the original activity. CONTRIBUTION No. 1929 FROM THE Identification of Interconversion and Degradation ProdGATESAND CRELLINLABORATORIES OF CHEMISTRY ucts.-Paper chromatography of the mixed sugar fraction CALIFORNIA INSTITUTE O F TECHNOLOGY in the 80% phenol-water system revealed three components PASADENA 4, CALIFORNIA when sprayed with the normal carbohydrate reagents such as aniline phthalate and 3,54initrosalicylic acid (Rr 0.33, 0.41 and 0.50). However, when ammoniacal silver nitrate used, a fourth component of low R, value (Rr 0.16) also Epimerization and Fragmentation of Glucose by was slowly appeared. Radioautographs of these chromatoQuaternary Ammonium Base Type Anion Ex- grams showed all four spots to be radioactive. Three of these components (Rt 0.33, 0.41 and 0.60) were identified change Resins' by cochromatography as glucose, fructose and mannose, BY DONALD R. BUHLER,RICHARD C. THOMAS, B. E. CHRIS- respectively. The fourth component is as yet unidentified. TENSEN A N D CHIHH. WANG Paper chromatography of the mixed acid fraction using a pentanol-formic acid-water system and a multiple-spray AUGUST11, 1954 RECEIVED developing techniques revealed six components ( RI 0.08, The interconversion and degradation of mono- 0.15, 0.23, 0.35, 0.56 and 0.74). Radioautography, however, indicated only three of these were radioactive (Rf and disaccharides through the action of anion ex- 0.35, 0.56 and 0.74). These were identified, by color rechange resin (quaternary ammonium base type) in actions with the various spray reagents and co-chromatogits hydroxyl form has been reported recently by raphy with authentic samples, to be: lactic acid (major several worker^.^-^ By analogy one would expect constituent), glycolic acid and lactyllactic acid. The compound presumably results from the autoesterifithe mechanism of these reactions to be similar to latter cation of lactic acid during concentration of the samples for that of the interaction of saccharides and strong paper chromatography.636 The other three non-radioactive alkali ; however, failure to find mannose in the in- components have not yet been identified. Isolation of Acids.-Half of the mixed acid fraction was terconversion products has been cited by Rebenfeld to silica gel column chromatography according to and Pacsu4as evidence that the ene-diol mechanism subjected the procedure of Bulen, Varner and Burell.? Three peaks for the Lobry du Bruyn transformation is not in- were observed: a large radioactive lactic acid band convolved. I n the case of the degradation products, taining essentially all of the starting activity, a very small five acidic compounds have been detected, but radioactive glycolic acid band and a small non-radioactive band. The purity and identity of all fractions were estabonly two have been identified, these being lactic lished by paper Chromatography. The unknown non-radioand glycolic acids.2.a The mechanism of the for- active acid component could not be detected readily on mation of these acids has not been investigated thor- paper chromatograms and it is as yet unidentified. The glycolic acid content of the combined glycolic acid oughly. fractions was assayed colorimetrically t o be 0.95 mg. acIn the present study, mechanism of the intercon- cording t o the procedure of Newburgh and B u r r k 8 After version and degradation of glucose through the ac- addition of the carrier, the diluted glycolic acid was isotion of Amberlite IRA-400, a quaternary ammo- lated as its calcium salt. The lactic acid was isolated from nium base type resin, was probed by the use of ra- the corresponding fractions as the zinc salt without dilution; dioactive glucose-2-C14. The interconversion prod- yield 41.5 mg. 211lactate trihydrate. Degradation of Glycolic and Lactic Acids.-The glycolic ucts were examined by means of chromatography acid was oxidized with lead tetraacetate according to a modiand radioautography. Lactic and glycolic acids fication of the procedure of Schou, et al.9 To a frozen mixwere isolated from the degradation products and ture of calcium glycolate in glacial acetic acid was added
-
(1) This research was supported by contract No. AT(45-1)-573 from The Atomic Energy Commission. Published with the approval of the Monographs Publications Committee, Research Paper No. 256, School of Science, Department of Chemistry. Presented before the Northwest Regional Meeting of the American Chemical Society, Richland, Washington, June, 1954. (2) A. C. Hulme, Nature, 171, 610 (1953). (3) J. D. Phillips and A. Pollard, ibid., lT1, 41 (1953). (4) L. Rebenfeld and E. Pacsu, THIS JOURNAL, 16, 4370 (1953).
( 5 ) M. L. Buch, R. Montgomery and W. L. Porter, Anal. Cham., 84, 489 (1952). ( 6 ) M. Ohara and Y . Suzuki, Science ( J a p a n ) , 81, 362 (1951). (7) W.A. Bulen, J. E. Varner and R. C. Burrell, A n a l . Chcm.. 84, 187 (1952). ( 8 ) R. W. Newburgh and R. H. Burris, Arch. Biochem. Biophys., 49, 98 (1954). ( 9 ) L. Schou, A. A. Benson, J. A, Basshamand M. Calvin, Phyriologis Planlarum, 3, 487 (1950).
