NOTES
June, 1961 C1
+
1085
NOCl +NO C1 NOCl +NO Clz
+
+
The rate constants for the two steps are 10l2exp(-38000/RT) set.-' and 1013 exp(-llOO/RT) mole-’ cc. sec. -I, respectively. According to this mechanism the half-life for the decomposition of NOCl under our conditions would be about 10I6 seconds. It is clear that the rapid isotope exchange reaction which we have observed cannot take place by a rate-determining decomposition of l4NOCI followed by a rapid reaction of 15N0with C1 or Clz, since the rate of decomposition of KOCl by either the bimolecular or unimolecular is much too slow. It seems highly likely that the isotope exchange reaction involves the attack of W O upon l4NOCI. We can picture this reaction as taking place in either of two ways: The first possibility is a displacement reaction on chlorine in which the transition state may be pictured as 0 “N--Cl-
14N0
0.8
0.6
0.2
The second possibility is the formation of a shortlived intermediate which rearranges to give the O~~N-CI
I
1
OW
5.5
5.6
5.7
exchanged products. The new bond that is formed 1,P. in the intermediate involves the unshared pair of Fig. 1.-(1) 14NOCl; (2) 1sNOC1, pressure 10 mm., cell length 20 mm. electrons of the nitrogen of NOCl being donated fied material showed that it was essentially free of the two to the NO. If the first possibility is correct then one should find a similar bimolecular reaction major impurities, HC1 and NOz. The mea were introduced into a 5-cm. infrared cell with Calf windows at the between NO and NOG1 (first-order in NO and first- desired pressure using a conventional vacuum line. The order in N02C1) to give NOz and NOC1. If, on mercury of the manometer was rotected from the NOCl the other hand, the second possibility is correct by a layer of perfluorokerosene. &he spectra were obtained one would not expect to find a reaction between with a Perkin-Elmer Model 21 Spectrometer equipped with NO and NO&l whose rate was dependent upon the a CaF2prism. pressure of KO since NOzCl does not have an unACIDITY COXSTANT OF A PROTEIN shared pair of electrons and hence is incapable of forming the postulated intermediate. CONJUGATE I N DzO A rapid reaction6 does in fact, occur between NO BY W.-Y. WEN AND I. M. KLOTZ and NOzCl to give NO2 and NOCl which is first Department of Chemistry, Northwestern Univermty, Evanslon, Illinois order in both NO and NO2CI, whose rate constant Received October 98, 1960 is 0.8 X 1Ol2 exp(-6900/RT) mole-l cc. sec.-l. This reaction has a half-life of several seconds The relative strengths in solution of hydrogen under our conditions of temperature and pressure. bonds involving protons and deuterium atoms, Thus we conclude that the isotope exchange respectively, have been the subject of numerous reaction between 15N0 and 14NOCl and the re- investigations. 1-6 For isolated molecules in the action between NO and N02C1 proceed by a dis- gaseous state, heats of dimerization’ show clearly placement on chlorine and not via the formation that bonding by deuterium is stronger, but only of the intermediate pictured above. It is interest- slightly so. For solutes in solution, however, there ing to note that whereas this reaction takes place are competing effects between substituents of the via a displacement on chlorine, the analogous re- solute and the solvent, and the net effect on physicoaction involving nitrite esters does not take place chemical behavior of the solute is not easy to previa a displacement on oxygen. Thus we have dict. another example of the frequentlyobserved phenomWe have r e ~ e n t l y ~interpreted .~ the shifts in enon that free radical reactions occur readily by (1) S. Korman and V. K. LaMer, J. A m . Chem. SOC., 68, 1396 displacement a t a univalent atom (halogen or hy- (1936). drogen) but not a t a polyvalent atom. (2) F. C. Nachod, 2. physik. Chem., A182, 193 (1938). Experimental 16NO was made in the manner previously described.* l4NOCI was purchased from the Matheson Com any and purified by distillation.B The infrared spectrum o?the puri( 5 ) E. Freiling, H. Johnston and R. Ogg, J . Chem. Phys., 8 0 , 327
(1952).
( 6 ) “Inorganic Syntheses,” McGraw Hill Book Co., New York, N. Y.. 1939, p . 55.
(3) A. H. Cockett and A. Ferguson, Phil. Mag., 88, 693 (1939). (4) F. A. Long and D. Watson, J. Chem. Soc., 2019 (5) M. Calvin, J. Hermans. Jr., and H. A. Scheraga, J. A m . Chem. Soc., 81, 5048 (6) G. Dahlgren, Jr., and F. A. Long, ibid., 88, 1303 (1960). (7) A. E. Potter, Jr., P. Bender and H. L. Ritter, J. Phys. Chem., 69. 250 (1955). (8) I. M. Klotz, Science, 198, 815 (9) I. M. Klotr and H. A. Fiess, Biochim. st Biophya. Acto, 8 8 , 57
(1958).
(1959).
(1958).
(1960).
NOTES
1086
Vol. 65
acidity constants of groups conjugated to proteins in terms of a crystallization of water around certain side chains of the macromolecule. It seemed of interest. therefore, to see whether any difference could be observed for hydration by deuterium oxide as compared to normal water. For this purpose we I have measured the pK, of dimethylaminonaph$02-R Son-R thalenesulfonyl chloride bound (separately) to (BHI-1 (B) protein and to a reference small molecule in D20 and a corresponding equation in which D + disand H20, respectively. sociates instead of H+. Experimental Materials.-G-ystalline bovine serum albumin was purchased from Armour and Co. The l-dimethylaminonaphthalene-5-sulfonyl chloride was a purified product of the California Corporat,ion for Biochemical Research. Heavy water was obtained from the Stuart Oxygen Co. and warranted to be 99.ii70 DzO. Preparation of Conjugates.-To a solution of 6.7 X M serum albumin in 0.1 M KaHCOs was added, a t 0 O , a ten-fold molar excess of dimethylaminonaphthalenesulfonyl chloride, in acetone solution at a concentration of 0.007 M. The mixture m-as stirred in the cold for 21 hours and then passed through an anion-exchange resin (IR.4-400) to remove any hydrolyzed uncoupled dye. The effluent was dialyzed against water in the cold for two days to remove acetone and salt. The final outside solution showed no fluorescence, whereas the solution inside the cellophane casing showed strong fluorescence under an ultraviolet lamp with 360 mp radiation. The conjugated protein was isolated by lyophilization. Absorbance measurements at 341 mp with a sample of this material in aqueous solution at pH 9 indicated 7.0 moles bound dye per mole protein, if \ye awume a molar absorptivity of 3.36 X lo3 1. mole-' cm.-' as found by Hartley and MasseylO for the chymotrypsin-c.ye conjugate. The glycine-dye conjugate was the same sample as that used previously.9 Its molar absorptivity in DzO was 4.43 X lo3 1. mole-' ern.-', not significantly different from the value reportedlo in HzO,4.55 X lo3. Titration Procedure.-The solid conjugate with protein or glycine was dissolved in H 2 0 and D 2 0 , respectively, to give a solution with an absorbance of 0.3-0.4 near 340 mp. Dissolution of solute in D 2 0 was followed by a hydrogen-deuterium exchange reaction which lowers the purity of D20. However, since the protein concentration M, the decrease in DzO content used wah only about would not exce1.d 0.1% and is, therefore, completely negligible. Iikem-isi?no correction was applied for the exchange reaction caused by the addition of minute quantities of HC1 or KaOH used to change the p D of the solution. The pH's (or p D ' s ) of the solutions were measured a i t h a Beckman pH meter, model G. In a DzO solution pD may be computed from the empirical equation of Glasoe and Long11 pr)
=
pH-meter reading
+ 0.40
(1)
where the pH-meter reading is obtained with a glass electrode-calomel electrode combination standardized to read pH in,H20solutions. Absorption spectra were obtained with a Beckman DU spectrophotometer at about 25'. For each soliltion of conjugate the spectruni was measured over a sei+ies of pH's as described previouslyg until enough data had been accumulated to provide a smooth spectrophotometric titration curve.
Results and Discussion The acid-base equilibria being studied may be represented by the equation (10) B
9 . Hartley and V. hIassey. Bcochzn. et Bzophys. Acta, 21,
58 (19.56).
(11) P. K. Glasoe and F. A . Long, J . Phys. Chem., 64, 1%
(1960).
TABLE I x(CHdZ ACID-BASEEQUILIBRIA OF
0;. IN
/
WATERAT 25"
/
S0z-R Nature of R
Glycine Bovine serum albumin a Taken from ref. 9.
p K H in Hz0 p K D in Dz0
3.99" 1.47
4.47 1.84
ApK
0 48 0 38
When R represents a small molecule, in our case a glycine residue, the dissociation can be described by the equation (3)
or by (4)
pK's can be evaluated readily by simple graphical method^.^ When R is a large protein molecule, for example bovine serum albumin, equations 3 and 4 no longer adequately correlate the ionization equilibria. Nevertheless, since we are interested in the present context merely in a comparison of behavior in HzO and DzO, we may define pK for the dyeprotein conjugate as that pH at which (B) = (BH+) or that pD at which (B) = (BD +). Acidity constants so calculated are assembled in Table I. The shift in pK of the dye as one goes from glycine to the macromolecular protein is large in either solvent, H 2 0 or DzO, being 2.52 units in the former and 2.62 in the 1at)ter liquid. It is clear then that both solvents produce a marked masking of the titrated dimethylamiiio group. The difference in masking effect between the tu-o solvents is only 0.10 unit, a quantity which is not larger than the combined uncertainties in the determiiiatioii of the various pK's involved. We conclude, therefore, that diff ereiices between hydrogen-bonding strengths in liquid water versus hydration water of proteins, are not appreciably different in DzO aq compared to HzO. Acknowledgment.-This investigation was carried out with the aid of a grant from the National Science Foundation.
