WILLIAMCOHENAND BERNARD F. ERLANGER
3928
acetate (19.90 g., 0.2 mole) and 0,O-diethyl phosphorochloridothioate (37.8 g., 0.2 mole) were stirred a t 75" for 5 hours. The mixture was cooled t o room temperature and filtered. Evaporation of the solvent left 42.4 g. of yellow
[CONTRIEUTIOS FROM
THE
Vol. 82
oil, which was distilled at 0.75 mm., t o give 8.20 g. of recovered 0,O-diethyl phosphorochloridothioate, b.p. 73-78". The pot residue thickened to a viscous, undistillable t a r during the distillation.
DEPARTMESTOF MICROBIOLOGY, COLLEGEOF PHYSICIANS ASD SURGEOSS, COLUMI~IA USIVERSITY, S E T V YORK, NEW YORK]
Studies on the Reactivation of Diethylphosphorylchymotrypsinl BY WILLIAXCOHENAND BERNARD F. ERLANGER RECEIVED DECEMBER 28, 1959 The reactivation of a lyophilized preparation of diethylphosphoryl ( D E P ) chymotrypsin by approximately forty nucleophilic agents (oximes and hydroxamic acids) was examined, using a rapid colorimetric assay for chymotrypsin. The !iydroxamic acids tested were synthesized by the usual methods as well as by two new synthetic techniques. With the exception of three compounds, all of the nucleophilic agents possessed activities which were related to their dissociation constants. T h e three exceptions, N-phenylbenzohydroxamic acid, N-phenylnicotinohydroxamicacid and pyridine 4-aldoxime dodeciodide, showed enhanced activities. The effect of pH on the reactivation process was also studied as well as the stability of DEP-chymotrypsin relative to chymotrypsin. Kinetic data were calculated on the basis of the catenary reaction DEP-chymotrypsin + chymotrypsin +. denatured chymotrypsin. Preparations of DEP-chymotrypsin could be completely reactivated even after standing several months.
Introduction
We are reporting the results of a study of the reactivation of lyophilized preparations of TEPPa-Chymotrypsin reacts stoichiometrically with inactivated chymotrypsin by a series of oximes certain organic phosphate^,^,^ such as tetra- and hydroxamic acids. In order to facilitate this ethylpyrophosphate (TEPP) ' and diisopropyl study, a rapid assay method for chymotrypsin has fluorophosphate (DFP), with a resulting loss of been developed, which has made possible the screenenzymic activity. Considerable experimental evi- ing of more than 40 compounds. Furthermore, dence points to a phosphorylation of the hydroxyl two new methods have been devised for the syngroup of a serine residue located a t the active site thesis of hydroxamic acids directly from carboxylic of the e n ~ y r n e . A ~ number of other esterasesjS6 acids using, in one case, phosphorus pentoxide as and phosphoglucomutase7 are similarly affected. the condensing agent, and, in the other, a mixed In the case of DFP-inactivated cholinesterase, anhydride intermediate for the preparation of Wilson and were able to design effective hexano- and N-phenylnicotinohydroxamic acids, nucleophilic reactivators as a result of a careful respectively. study of the specificity of the enzyme. Wilson Experimental also recently reported that pyridine aldoximes Enzymes .-Three times crystallized a-chymotrypsin which could slowly reactivate chymotrypsin inhibited been dialyzed salt-free and lyophilized was purchased by TEPP.IO Earlier work by Cunningham has es- had from Worthington Biochemical Co., Freehold, New Jersey. tablished that chymotrypsin, inactivated by diDiethylphospl-ioryl-o-chymotrypsin ( DEP-chymotrypsin) ethyl p-nitrophenylphosphate," can be partially was prepared by the method of Jansen, et aZ.2 The dialyzed reactivated by hydroxylamine, and while this paper and lyophilized material retained 0.1-0.7% of the initial acwas in preparation, Green and Nicholls reported tivity. Nucleophilic Reagents.--% number of oximes and hythe reactivation of Sarin-inactivated chymotrypsin droxamic acids were purchased from Distillation Products Industries, Rochester 3 , Xew York: acetone oxime, benzby several oximes and hydroxamic acids.12 (1) This work was supported in part by research grant E-1672 from t h e National Institute of Allergy and Infectious Diseases, National Institutes of Health and a contract wlth the Office of Naval Research (NOnr 286(44)). A preliminary account appears 1n t h e Abstracts of t h e 136th American Chemical Society Meeting, Atlantic City, A-. J., September 1959, p. 59-C. (2) E. F.Jansen, A I . D. F. Nutting, R. Jang and A. K. Balls, J . B i d . Chenz., 179, 189 (1949). ( 3 ) J. H. Fleisher, R J. Jandorf, W . H. Summerson and D. D. Norton, Federalion P Y O C 9, . , 171 (1930). (4) X-. K . Schaffer. S. C . M a y . Jr., W. H. Summerson, J . B i d . Chew? , 202, 67 (1933). (5) N . K. Schaffer, S. C . S l a y , Jr., W. H . Summerson, i b i d . , 206, 201 (1954). (6) J. A. Cohen, R . A . Oosterbaan, lf,G. P. J . Warringa a n d 1%. S . J a n s z , D ~ S C U S SF~a rGa d~ a?y ~SOC..20, 114 ( 1 9 3 ) . ( 7 ) L. Anderson and G . R. Jnlles, A?.&. Biockrm. Biophys., 70, 121 (lY57). (8) I. B. R'ilson, F p d ~ i a l i ~Pi'oc.. n 18, 732 (1939). (!I) h.I,. G r e e n and H J . Smith, Eic~hcuz.J., 68, 32 (1928). ( I O ) I. B. \Tiison, S . Ginsburg and C. Quan. Auch. Biochcm. B i ~ p h Y s . , 77, 286 (1958). (11) L. W.Cunningham, Jr., .I. B i d Chela., 207, 443 (1954). (12) A . L. Green m d J . D Sicholls, Bio
