NOTES
Sept., 1959 tion of the alloocimene and the dimer at equilibrium a t 189.5 and 204.5'. The density of the equilibrium mixture a t 25", was 0.875 g. per ml. At 204.5", its density was found to be 0.74 g. per ml. The densities a t these latter temperatures were determined by the same procedure described in the case of alloocimene. A t 189.5' K =
/
[Dimer] - 0.89( 0.75)lOOO [Alloocimene]2 272
A t 204.5", K =
272
5.6 l./mole k, =
l8
5.6
=
3.2 X 10-4 min,-l
The energies of activation for the forward and reverse reactions and the heat of the reaction were calculated from the values of k4, k g and R a t the two temperatures. These values are listed in Table 111. TABLE I11 AH AEaat (oal. mole-l (cal. mole-' deg. -1) deg. -1)
Reaction
2 Alloocimene 4 Dimer Dimer -+ 2 Alloocimene
-5300 +5300
22,000 28,000
A&,t
(e.u.)
- 13.6 - 2.5
The entropies of activation for the forward and reverse reactions were estimated by solving these equations for ASa. k =
RT e AH~/RT~AS&/R Nh
where k is k4 or kS as the case may be. These values also are listed in Table 111.
THE B1'NUCLEAR MAGNETlC RESONANCE CHEMICAL SHIFTS AND SPIN COUPLING VALUES FOR VARIOUS COMPOUNDS BY THOMAS P. OSAK,HERBERTLANDESMAN, ROBERTE. WILLIAMS A N D I. SHAPIRO~ Research Laboratoru, Olin Mathieson Chemical Corporation, Pasadena, California Received February 84, 1969
1533
TABLE I B'1 N.M.R. CHEMICAL SHIFTS AND SPINCOUPLING VALUES Compound BrHd (apex boron) (CSz soln.) B6H11 (apex boron)' B6H9 (apex boron) B6HlO (apex boron)d BdHio (BH)' NaB& (aq. s o h ) LiBH4 (ether soln.) BsHsBr (apex boron) (CSz soln.) BIOH14 (2,4 pos.) (CSz s o h ) (CHs)zHNBHa (benzene soln.) BSHB(base borons) BsHsBr (base borons) (CSz soln.) BsHsI (base borons) (CSz s o h ) CsHsNBHa (pure 1iquid)u (CH3)sNBHs (benzene soln.) NaB(C6Hs)a (as. s o h . ) B4Hm (BHz)' €313 (liquid melt) &HI1 (base B-H IC NaBFr ( a s . soln.) BFa.piperidine (CSz solnJh NH4BF4 (aq. s o h ) BFa~hexamethylenetetrarnine RFs.O(n-CaHe)z BFa*O(CzHs)z HBF4 (50% aq. soln.) BFs.P(CeHs)s (CHC13 s o h ) BFa.S(CzH4-CeHs)a (CHCla s o h ) (5,7,8,10pos.) (CSe s o h ) BIOHI~ Tetraacetyl diborate (CHCla soln.) NaBOz (B(OH)4-) (aq. s o h ) NaBsOs (aq. s o h ) (see text) &Hll (base BHz)' LiB(0CHa)r (methanol soln.) NaB03 (aq. s o h ) KzB407 (as. soln.) HBCla.O(CzHs)a DBClz.O(CzHs)z NazBdO, (as. soln.) BFa (gas) (NH4)aBao~(aq. s o h ) BClvO(CaHs)z (ether s o h ) N(CHzCHz0)3B (aq. soh.)! BlOHlP (1,3,6,9pos.) (CSz soln.) KBsOs (aq. s o h ) Tri-o-chlorophenyl borate (ether soh) NaBsOs (aq. s o h ) (see text) BaHm (base borondd Tri-o-cresyl borate (ether s o h ) BaH6 (gadk Methyl metaborate (benzene soln.) n-Butyl metaborate (benzene soln.) B(OCHzCH=CHa)a B(0CHa)s B(0CzHs) s B(0H)r (as. s o h ) B (0CzHs)zCI HB(0CHs)z DB(0CHa)z B(OH)z(n-CeHm) (ether s o h ) B (0CzH.d CIP BZH203m BBrs BCla R(CZH&)3 . .
J ,C . / B .
6a
5 5 . Ob 53.5 51.8 51.2 40.0 38.7 38.2 36.4 34.9 15.1 12.5 12.5 11.8 11.5 9.1 8.2 6.5 5.5 2 . 3 est. 2.3 2.3 1.8 1.4 0.0 0.0 -0.1 -0.4 - .5 - . 5 est. -1.1 1.0 -1.3 -1.3 f 1 0 - 2 . 9 eat. -2.9 -5.5 -7.5 f 1.0 -7.9 -8.0 -8.9 -9.4 f 1.0 -10.3 -10.5 -10.7 - 1 2 . 4 est. -13.0
*
-13.7 f 2 0 -14.4 f 1 . 0 -15.0 -15.0 f 1.0 -16.6 -17.3 -17.5 -17.5 -18.11 -18.1 -18.8 f 1.0 -23.3 f 1 . 0 -26.1 -26.7 -29.3 f 1 0 -32.5 f 1 0 - 3 3 . 6 f 3.0 -40. I t -47.71 -85 f 1 . 0
* *
170 (BH) 5 173 (BH) 5 182 (BH) f 5 154 (BH) f 5 82 ( B H ~ ' f 3 75 (BH4) 3~ 3
..........
*
158 (BH) 5 91 (BH3) 3 160 (BH) 1 161 (BH) f 5 100 (BH) 5 90 (BHa) f 3 101 (BHa) zt 3
* * *
..........
123 (BHz)
*3
..........
133 (BH) est
.......... .......... ..........
.......... .......... .......... 141 (BH) est.
.......... 130 (BHa) est.
.......... .......... ..........
152 (BH) f 5 ? (BD)*
..........
..........
..........
138 (BH) est.
.......... .......... 100 (BH) f 5
.......... 128 (BHz) =k 4
..........
.......... ..........
.......... ..........
.......... ..........
141 (BH) f 5 24 (BD) 3= 4
The BI1 nuclear magnetic resonance spectra of .......... a variety of boron containing compounds have .......... 169 (BH) f 5 been obtained with a Varian V-4300 high resolution .......... n.m.r. spectrometer operating a t 12.83 Me. ; .......... the chemical shifts and spin-spin coupling values2 .......... are given in Table I. Boron trifluoride ethyl a 6 = ( H . - H , ) / H , X 1 0 6 ; estimated deviation h 0 . 5 etherate has been selected a~ the zero reference4 unit unless otherwise noted. Pentaborane-9 was used as a because of the sharp resonance line as well as the secondary standard; spacings were measured with respect to (1) Box 24231, Loa Angeles 24, California. ( 2 ) I n addition to spin-spin coupling values of boron to hydrogen, the boron-deuterium coupling value of DB(0CHs)z was obtained
(see Table). Other deuterated boron compounds (ref. 3) fail to exhibit the B-D coupling due to broad resonance lines which overlap. (3) (a) R . E. Williams and I. Shapiro, J. Chem. Phys., 29, 677 (1958); (b) T. Onak, H. Landesman and I. Shapiro, THISJOURNAL, 62, 1605 (1958). (4) W. C. Diokinson, Phys. Rev., 81, 717 (1951).
the low field doublet (160 c./s.). This is a t slightly higher field than that portrayed by R. Schaeffer, J. N. Shoolery and R. Jones (ref. 11). c R . E. Williams, S. G. Gibbins and I. Shapiro, J. Chem. Phys., 30, 320 (1955). d R. E. Williams, S. G. Gibbins and I. Shapiro, ibid., 30, 333 (1959). e R. E. Williams, S. G. Gibbins and I. Shapiro, Abstracts of the 135th Meeting of the American Chemical Society, Boston, Mass., April 1959, 38M. f Agrees with J-value given by R. Ogg, J . Chem. Phys., 22,1933 (1954). In benzene 8 = 12.3, J = 98. In neohexane 6 = 1.8. Line width smears B-
NOTES
1534
Vol. 63
D spin-spin coupling (ref. 3).
j Triethanolamine borate (ref. 5) in chloroform 8 = -11.2. * R. Og , (Table I, footnote f) report J = 125; see also J. N:Ehoolery, Faraday SOC.Disc., 19, 215 (1955). ‘Dickinson (ref. 4) reports 8-values: B(OCHa)!, -18; BBra, -44; BCls, -46 J. F. Ditter and I. Shapuo, J. Am. Chem. Soc., 81, 1022 (1959).
The few series of boron compounds listed in Table I are essentially in agreement with the polar bond theory which requires that shifts become more negative as the electronegativity of the substituents increases. A trend toward lower field with increased electronegativity of the ittom bonded to ready availability of the etherate. The compounds boron is noted in the trihalide series B13, BRr3 were studied as liquids or in solution. Where the and BC13.’s8 Increased ionic character of BF34,9s10 line width of the B” resonance was broadened by may be responsible for its deviation from this the viscosity of the solution, an inert solvent was pattern. The inductive effect of the halides upon added to reduce the viscosity, narrow the line the B” chemical shifts is again evidenced in the width and thus obtain a measurable signal. Solvent substituted pentaboranesl‘ where the apex boron effects upon the chemical shifts of Bll resonance of bromopentaborane is a t lower field than the spectra appear to be negligible a t 12.83 Mc. apex boron of iodopentaborane. except in those cases where a definite chemical The chemical shift values of certain tetravalent reaction has occurred. Therefore, chemical shift boron ions are in the order BH4->B(CsHa)4-> values for boron compounds can be regarded as B(OCH3)4-. Similar trends are observed in the characteristic empirical constants for identification substituted phosphorus compounds, ‘viz., PHI> purposes. In addition, it is possible to deduce P(C~HS)~>P(OCH&,*~ and in the methyl proton from these values information about the bonds spectra of the related carbon compounds, viz., within the molecules studied. CH4>CH3-CeH,>CH3-OR. l 2 The relative shieldWithin a series of planar boron compounds a ing by the adjacent elements is H>Caromatic> general shift to higher field occurs with increasing 0 which is inversely proportional to the electrodouble bond character between boron and its negativity of the adjacent atoms as expected. adjacent atoms. Triethylboron, positioned a t Structural information about borates in solution lowest field, represents the only compound in is suggested by the following observations: two Table I which cannot have more than a sextet of peaks are seen in the spectrum of an aqueous soluelectrons around the boron atom. The atoms tion of sodium pentaborate in contrast to one peak adjacent to the boron in the remaining trisubsti- in an aqueous solution of potassium pentaborate. tuted compounds are capable of contributing A synthetic solution prepared from sodium hyelectrons to the boron, thus enhancing the double droxide and boric acid with the sodium pentabond character and approaching an octet of elec- borate B/Na ratio also produces two peaks. Addition of acid to the sodium pentaborate solution trons around the boron atom. The tetravalent and trivalent chemical shift decreases the intensity of the high field peak (6 regions can be subdivided further on the basis of 1.3) while shifting the low field peak a t 6 - 14.4 individual inductive effects of the atoms directly to 6 - 18.8 (boric acid). Addition of small bonded to boron since non-adjacent atoms have amounts of base to the pentaborate solution also little effect on the shielding. For example, chem- decreases the intensity of the high field peak (6 ical shift values from Table I indicate that 1.3) but shifts the low field peak (6 - 14.4) to three oxygens bonded to trivalent boron occur higher field. These observations would indicate approximately in the region 6 - 14 to - 19. An that the low field peak which shifts with acid and exception is triethanolamine b ~ r a t e which, ,~ when base additions represents an equilibrium mixture compared to other alkyl borates, is found a t higher of trivalent boric acid and tetravalent monoborate field. This shift is interpreted as increased shield- ionla; the appearance and disappearance of the ing around the boron atom due to some B-N high field peak shows another borate species is bonding, thus increasing the tetrahedral character present, possibly pentaborate or tetraborate ion. and number of bonding electrons around the boron A solution of potassium pentaborate, on the other atom (see above). Another exception is the oc- hand, has only one peak which shifts to higher field currence of the tetraacetyl diborate resonance line with addition of base. Low solubility of an interat higher field (6 1.1) than expected for a boron mediate potassium polyborate may explain the bonded to three oxygens. The tetraacetyl di- lack of a high field peak such as was present in the borate resonance line would be expected to occur sodium pentaborate spectrum. Addition of mannitol to boric acid (6 - 18.8) a t slightly lower field than the alkyl borates if only an inductive effect were operating. However, (7) The chemical shift of BClr was found in the same position in tetraacetyl diborate may undergo chelation to both liquid and vapor phase. give either four-membered or six-membered rings16 (8) For similar shifts in other element halides see: (a) P. C. Lauterwhich would increase the tetrahedral character bur, J . Chem. Phus., 26, 217 (1957); (b) C . H. Holm, ibid., 26, 707 (1957): ( 0 ) N. Muller, P. C. Lauterbur and J. Goldenson, J . Am. Chsm. (see above) and the shielding of the boron atoms. BOG., (1956): (d) J. R. Van Wazer, C . F. Callis, J. N. Shoolery It is observed that the tetraacetyl diborate reso- and R.7 8C., 3557 Jones, ibid., 78,5715 (1956). nanceline (6 - 1.1) occursin the region characteristic (9) Linw Pauling, “The Nature of the Chemical Bond,” Cornell of the tetrahedral boron atom bonded to four Univ. Press, Ithaca. N. Y., 1948, second edition, p. 239. (IO) R. A. Ogg, Stanford Univ., Technical Research Report, MCCoxygen atoms (e.g., B(OH)4-, 6 - 1.3; LiB(OCH3)4, 1023-TR-120, January, 1955. 6 2.9). ( 1 1 ) R. Schaeffer, J. N. Shoolery and R. Jones, J . A m . Chem. XOC.,
-
-
(5) 2,8,9-Trioxa-5-aza-1-borabicyclo [3.3.3]undecane in the oxa-azabora system of nomenclature. (6) L. A. Duncanson, W. Gerrard, M. F. Lappert, H. Pyszora and R. Shafferman, J . Cham. Xoc., 3652 (1958).
80, 2670 (1958).
(12) N. F. Chamberlain, Anal. Chem., 32, 56 (1959). (13) Peter H. Kemp, “The Chemistry of Borates,” W. 8. Cowell Ltd., Butter Market, Ipswioh, 1956, pp. 55-64.
NOTES
Sept., 1959 did not shift the resonance line, but addition of excess alkali to this solution shifted the resonance line to 6 - 7. These observations are in agreement with equilibrium constant^'^-'^ for this system which indicate that only in basic solution is appreciable mannitol-boric acid complex present. Nuclear magnetic resonance B l1 chemical shifts also may be used to identify products and monitor the course of reactions without removing the materials from the reaction medium. For example, BClzOEt (6 - 32.5) in contact with ether quickly disproportionate^^^^'^ to BCl3:OEtz (6 - 10.5) and BCl(OEt)2 (6 - 23.3) both of which are detected by n.m.r. I n addition, the subsequent slow cleavage of BC13:OEtzis observed by the intensity decrease a t 6 - 10.5 arid intensity increase a t 6 - 23.3. (14) A . Deutsch and 9. Osoling, J . A m . Chem. Soc., 71,1637 (1949). (15) 8. D. Ross and A . J. Catotti, ibid., 7 1 , 3563 (1949). (16) €1. Ramser and E. Wiberg, Ber., 63, 1136 (1930). (17) E. Wiberg and W. Sutterlin, 2. anoro. allgem. Chem., 202, 21
(1931).
THE DENATURATION OF PEPSIN. V. T H E ELECTROSTATIC FREE ENERGY OF NATIVE AND DENATURED PEPSIN' BY HAROLD EDELHOCH~ Conlrzbution from the Department of Pathologw and Onculogy, Kansas University Medzcal School, Kansas Catg, Kansas Receaved fifarch I d , 1969
It has been shown by viscosity, sedimentation and diffusion measurements that denatured pepsin has a much less compact structure than native p e p ~ i n . ~I n this report, this difference in structure is shown to markedly affect the electrostatic free energy of native and denatured pepsin. Methods.-A stock solution was prepared by dissolving native pepsin (Worthington Biochemical Corp.) in 0.001 M NaCl fpH -3.5) and then carefully adjusting, a t O", with dilute base to pH 6.65. This solution is quite stable at 24", the temperature of the experiments. Denatured pepsin solutions were obtained by raising the pII of the stock solution from 6.65 to 9.60 with base and then reacidifying to pH 6.65. Pepsin solutions were titrated with small volumes of concentrated solutions of salt in order t o keep the total volume approximately constant. The pH was read after each addition of salt. Other procedural details have been reported elsewhere .a14
Results and Discussion I n Fig. 1 is reported the effects of NaCl on the pH of native and denatured pepsin as well as the influence of BaCh on the pH of native pepsin. The difference in behavior of native and denatured pepsin to NaCl was rather striking. The origin of these differences must be related to the structure of the two forms of pepsin since their composition was identical. Moreover, the net negative charge of denatured pepsin a t pH 6.65 was greater than that of the native enzyme by at least the iiumber of carboxyl hydrogen-bonded groups (5 or 6 ) which are lost on d e n a t ~ r a t i o n . ~ The larger charge should therefore increase the electrostatic effects if other parameters are unaffected. (1) Supported in part by an Institutional Grant from the American Cancer Society and by Grants No. C-1974 and R G 4690 of the National Institutes of Health. (2) National Institutes of Health, Bethesda, Md. (3) H. Edelhoch, J . A m . Chem. Sac., 79,6100 (1957). (4) H. Edelhoch, abad., 8 0 , 6640 (1958).
1535
0
1 .O 2.0 - log IONIC STRENGTH.
3.0
Fig. 1.-The effect of NaCl on the pH of solutions of native and denatured pepsin; effect of BaClz on the p H of native pepsin; pepsin concentration = 5 mg./ml.
The theoretical expression derived by Linderstr@m-Lang6has been found to fit satisfactorily the titration curves of numerous proteins. It was assumed that all the members of each type of dissociable group possessed the same intrinsic dissociation constant pKo and that all charged groups were uniformly distributed on the surface of a sphere and that there were no interactions amongst these groups other than electrostatic. The relation may be written as pH
Ti - log ----i = (pKo)i - 0.8680Z
(1)
where ni is the total number of each type of dissociable group and ri is the number dissociated a t a particular pH value. 2 is the net charge on the protein and w is related to the electrostatic work required to remove a proton from a molecule possessing a charge of 2. The dependence of w on ionic strength and the radius is given in eq. 2 where R is the radius of the sphere, K, the reciprocal Debye radius, A , the exclusion radius, D, the dielectric constant, k, the Boltzmann constant,, e, the protonic charge and T the absolute temperature. A minimum value of R equal to 22 d. may be calculated from the molecular weight of pepsin (35,000) and an assumed partial specific volume of 0.75. An effective radius may also be calculated from the intrinsic viscosity (0.033)3by the Einstein equ:ition [v] = 1 0 NRe3/3M. ~ A value of Re S 26 A. was obtained in this way. This larger value is due to protein hydration or lack of spherical symmetry in native pepsin. Most likely both effects contribute in some degree. When w was calculated from equation 2 a t vnrious ionic strengths of NaCl and then plotted against the corresponding experimental value of p H , a linear relationship was observed above -0.01 I'/2 as seen in Fig. 2. The points increasingly deviated from a straight line as the ionic strength decreased below this value. When the pepsin gegenion concentration was included in the low ionic strength solutions, the discrepancy was reduced somewhat as shown by the arrows in Fig. 2.6 (5) K. LindersWm-Lang, Compt. rend. trau. Lab. Carlsberg, 15,
No. 7 (1924).
( G ) We need not concern ourselves greatly about the very low ionio strength data since the contribution of the charged protein-ion has been neglected and even more important, i t is doubtful whether the theory is
