RAGNAR ~STERBERG
2230
0
0-
GOz
+ SiI /I\
/I\
.Si
+
0
/I
c / \
+
02-
A A\ 0
the dangling 0- having been formed by radiolytic action on the SiOz. The new spectrum formed by addition of SO2 to irradiated Si02 is different from that formed by C02 and can only be attributed to 02-if the conditions of absorption of the latter are distinctly different in the two cases. I n contrast to the esr spectra which they yield when added to irradiated gel, both COzand SO2yield a singlet close to the free electron g value when they are present
on silica gel during irradiation (Figure 3). The G values determined from these spectra are at least an order of magnitude higher than for the esr signal in the absence of additive. The spectrum from GO2is similar in appearance to that assigned to GOz- in other media,21 but is observed at a somewhat higher g value. It is to be presumed that C02 and SO2 present during- irradiation form anions, as other electron scavengers are known to do. Xignals from Freshly Fractured Gel. The broad esr singlet (about 4 G between maximum and minimum) shown by SiOz, which has been powdered under vacuum, must be due to unpaired electrons on Si atoms or dangling SiO. bonds. The effect of NO in removing the signal is consistent with this conclusion. (21).An example and references are given in M. Shirom, R. F. C. Claridge, and J. E. Willard, J. Chem. Phys., 47, 286 (1967).
Proton Equilibria of Glycylglycylglycine and Glycylhistidylglycine in the Solvent 5 Volume
% Water in Methanol
by Ragnar Osterberg Department of Medical Biochemistry, Unhersity of Gbteborg, Gbteborg, Sweden
(Received October 23,1968)
The proton equilibria of glycylglycylglycine and glycylhistidylglycine were sLuc.Ledat 25” and 1.O M ionic strength by potentiometric titration in the solvent 5 vol % water in methanol by using a glass electrode. The upper ranges of concentrations (5 and 8 mM, respectively) were limited by the decrease in peptide solubilities in the regions of the isoelectric points. The effective water concentration was held constant during the titrations by generating OH- ions by constant current electrolysis. The results show a marked increase of 1.9-2.3 units in the pK values of the carboxyls when water (1.0 ;M NaC10J is replaced by the solvent methanolwater (1.0 iM NaC104). The pK values of the amino and imidazole groups, on the other hand, increase by only 0.2-0.4 unit.
Introduction Important aspects on the forces responsible for the tertiary structure of proteins have been advanced on the basis of solution data for low-molecular-weight Attention has been focused on the effect of water surrounding the protein molecules. Water seems to stabilize the structure of proteins by acting as an entropy source for the formation of hydrophobic bonds rather than by forming “icebergs” on the hydrophobic regions of the protein molecule.2 The acid-base properties of proteins also appear to be consistent with the view that their extensive hydration is due to reasons other than those which lead to “icebergs.” Most of the intrinsic pK values for proteins The Journal of Physical Chemistry
are of the same order of magnitude as the pK values of low-molecular-weight model compounds (see for instance Tanford’s reviewa). It must be kept in mind, however, that many proteins have but few groups whose pK values are markedly different from those of the same groups present in model compounds. The reason for this is not completely clear. The present study has been carried out in an effort to gain a more detailed understanding of the effect of the solvent water on the properties of acid-base groups (1) H. S. Frank and M. W. Evans, J. Chem. Phys., 13, 507 (1945). (2) W. Kaurmann, Advan. Protein Chem., 14, 1 (1959). (3) C. Tanford, ibid., 17, 69 (1962).
PROTON EQUILIBRIA OF PEPTIDES IN METHANOL-WATER present in proteins. I n particular, we are interested in such groups that are exposed to a medium having a lower dielectric constant, a lower local water concentration, and a more ordered water structure than pure water (cf. the discussion on the hydrophilic-hydrophobic regions of enzyme molecule^).^ As a model medium we have chosen 5 vol % water in methanol,6 which is reported to have these properties.’ This paper deals with the proton equilibria of two model compounds. For comparison the same compounds have also been studied in the solvent water. A constant and high concentration of background salt, 1.0 M NaC1O4, was used to maintain the activity factors constant.6 The liquid junction potential was reduced to a minimum by using almost the same medium in the salt bridge, the reference half-cell, and the measuring solution.’
Experimental Section In each titration H,the total concentration of protons, was varied by a progressive generation of OHions, while A , the total concentration of peptide, was kept constant The hydrogen ion concentration, h, was measured by the cell
I
I
-RE equilibrium solution G E
+
(1)
where GE is the glass electrode and R E the reference half-cell Ag, AgC110.99 M NaC104, 0.01 M NaCl,
5% H20-MeOH) 1.0 M XaC104, 5% H20-MeOH
(11)
The emf of cell T if defined as
E
=
Eo
+ 59.154 log h + E,
(1)
The OH- ions were generated at a platinum gauze immersed in the solution. The composition of the solutions and the apparatus were essentially the same as the circuit for constant current coulometry described by Biedermann and C i a ~ a t t a . ~ However, water was replaced by 5% water in methanol as the solvent. The titrations in water were carried out as previously described.1° The ionic medium was the same as that of the mixed solvent, 1.0 M NaC104. For low concentrations of peptide these titrations also employed generation of OH- ions by constant current electrolysis. For the titration in solvent 5, solutions were freshly prepared from weighed samples of peptide, crystallized as neutral “zwitterion.” Each sample was dissolved by adding appropriate amounts of HClO4 (1.0 M in (Na)C104) and 1.0 M NnC104, both solutions being in solvent S. Titrations were initiated immediately after this preparation to minimize the formation of the methyl esters. The upper limits of the peptide concentrations were the precipitation limits. Precipitation
2231
occurred in the isoelectric region at the concentrations 8 (glycylhistidylglycine) and 3 mM (triglycine) . We attempted to determine E,, the liquid junction potential, as a function of h in the solvent S by using the procedure described by Biedermann and SillBn.11 However, from repeated titrations it was found that the function ( E - 59.154 log h) could not be described as a linear function of h. The magnitude of E,, on the other hand, appeared to be much less than in water, so in our ranges, where -log h 2 2.5, we found E, 0.1 mV. The measurements commenced with a titration of Ho, the excess concentration of H+. From this portion of the titration we also determined Eo. The value of Ho was approximately known from analysis but was improved by a plot according to Gran.l2 The second portion of the titration gave the data ( H , E ) A . These data, in combination with Eo, were used to first calculate log h by eq 1 and then Y = (H - h)/A. An example titration is given in Table I. The potentials were constant within 5 min. The titrations were repeated to within 0.3 mV in the range -log h 6.5 and to 0.5-2 mV in the range -log h > 6.5. Magnetic stirring was used. The titration vessel was kept closed to avoid evaporation. After each titration, the contents were checked by weight. The coulometric method was standardized for the solvent S by titrations of standardized solutions of HC104, giving 0.1% agreements with the expected values, and by titration of 10 mM acetic acid in 0.5 M NaC104. For the latter we obtained -log h = 6.95 at Y = 0.5; Grenthe and Williams’ report 6.97. Apparatus. The potentiometric equipment has been previously described.lo All experiments in solvent S were carried out in a thermostated room at 25.0 f 0.1”. Burets and pipets were recalibrated for solvent S. The glass electrodes (Becltman No. 40498) were calibrated against a hydrogen electrode for the measured log h range both in water and in solvent S. The Ag, AgCl electrodes were made according to Brown.13 The
1). nuclear type, HqA7(@ The equilibrium constants were determined using the general (least-squares) minimizing computer program, Letagropvrid. l7 Preliminary constants were read off at Y = 1.5 and 0.5, for glycylhistidylglycine also at Y = 2.5. The computer searched for the minimum value of the sum
u = C(Yoalcd
- Yexptl)2
(2)
Starting from the preliminary constants the machine computed the quantity, Yoalod, either using the input, log h, pgl (r = l), or log h, A , pql (r > 1). It was also assumed that each titration had a constant error, 6Y. This error is treated as a constant (ks constant) by the computer and can be varied independently." Table I11 shows the pK values obtained, giving 3 u as limits. The systematic errors are indicated in Table 11, which also gives AY, the deviation of each point. To explain the deviation of the data for glycylhistidylglycine in solvent S we first introduced the dimers, HzAz and H4AZ2+. This gave U = 0.003 and c ( Y ) = 0.0076. Mononuclear species alone gave U = 0.005 and .(Y) = 0.009. When we also assumed a constant error, 6Y, we obtained for the mononuclear species U = 0.002, u(Y) = 0.0059, and 6Y values from -0.007 ( A = 8 mM) to +O.OlO (0.47 mM). The combination including the dimers gave U = 0.0018, .(Y) = 0.0059, and 6Y values from -0.0016 to +0.004. Although the lower 6Y values of this latter set support the occurrence of dimers, the error square sum and u(Y) are almost the same for
pK(Hz.4) (imidazole) .
I
PK(HA) (a-NHa *)
.
... 6.92 f 0.01 7.31 i.0.01
8.06f0.01 8.32 0.01 7.96 f 0.01 8.29 f 0.01
U(Y)
0.0035 0.0041
0.0058 0.0093
obtained by Letagrop.1'
both sets. Consequently, we cannot draw any valid conclusions regarding the possible presence of dimers. If they exist the logarithms of their constants, ppz,are (giving the maximum values within parentheses) Z 17.9 (
