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3188
degree of confinement or dielectric properties. Unlike the solvated halide ions, the solvation shell of the trapped electron cannot interchange solvent molecules with the bulk solvent. Consequently, the band positions do not reflect the bulk solvent composition. The orientation of 0-H bonds serves to differentiate in kind the electron trapped in an ethanol from a methyl-2-tet8rahydrofuran environment. Although no phase separation was evident, the mixtures might separate into domains of pure ether and ethanol character, in which case no specific difference in the kind of electron traps existing in rigid ethanol and methyl-2-tetrahydrofuran could be inferred. A mixture showing the near-infrared band, together with an intermediate absorption, showed the relative stability of the latter subject to heating (Figure 2). Electrons trapped in mixed ethanol and ether environments would be responsible for the intermediate optical (and e.s.r.) absorptions, which correspond to deeper potential traps than those provided by pure ether.
Acknowledgment. The author thanks Professor F. S. Dainton for helpful discussions and colleagues at the Cookridge Radiation Research Institute for experimental assistance. This work was done during tenure of a Brotherton Research Fellowship of the University of Leeds.
Refractive Index Dispersion in Equine Hemoglobin Solutions1
by W. H. Orttung and J. Warner Department of Chemistry, University of C a l i f w n k , Riverside, Californk 936132 (Received February 18, 1966)
Refractive index dispersion has been measured for several globular proteins in s o l u t i ~ n , ~but - ~ the available data on do not include wave length dependence. The data reported here were taken to complement Kerr effect optical dispersion studies,8 but appear i o be of some intrinsic interest, not only because regions of heme absorption are spanned, but also because hemoglobin is more a-helical t,han most other globular proteins. The refractive indices of dilute solutions of horse met- and oxyhemoglobin were measured in the 500-700-mp range using a BricePhoenix differential refractometerg modified by the addition of an Engis 505-01 monochromator and tungsten lamp. The contribution of the visible and nearultraviolet spectrum to An/c was calculated by the The J O U T ~of U ~Physical Chemistry
NOTES
Kramers-Kronig relation and subtracted from the data. The residual dispersion was normal, but considerably less than that of other nonheme globular proteins.
Experimental Hemoglobin was prepared from freshly drawn horse blood run directly into iced saline citrate solution to prevent clotting. The cells were spun out, then resuspended and washed five times with 1% NaCl. The packed cells were hemolyzed with 0.5 vol. of cold water and 0.4 vol. of cold toluene. The pH was brought to 6.8 by bubbling a 4 : l mixture of COz and 0 2 through the slurry and then crystallization was allowed to proceed overnight. Most of the cell bodies were separated by centrifugation and one washing with cold water. Two recrystallizations were carried out by raising the pH to 7.4 with 1 M K2HP04,adding a minimum amount of water to achieve solution, and centrifuging, with removal of more cell bodies. An equal volume of cold saturated (NH4)zS04 was then added for recrystallization. All steps were carried out at 0". Methemoglobin was prepared from oxyhemoglobin by ferricyanide oxidation according to Benesch, et al. ,lo and crystallized by dialysis against 2 parts of 4 M (NH*)ZS04 and 1 part of 2 M ("4)ZHP04. Concentrated stock solutions of both oxy- and methemoglobin were prepared and dialyzed extensively against cold distilled water, the concentrations then being determined by evaporation to dryness at 110". Dilutions of the stock solutions were made by weight with distilled water. Volume concentrations, c, in grams per milliliter, were calculated from weight fractions. The quality of the dilutions was monitored on a Cary 14 spectrophotometer. Calibration of the refractometer at 25" was carried out in a manner similar to that described previou~ly.'~ (1) This investigation was supported in part by Public Health Service Research Grant GM11683-01 from the Division of General Medical Sciences. (2) G. E. Perlmann and L. G. Longsworth. J. Am. Chem. SOC.,70, 2719 (1948). (3) M. Halwer, C. G. Nutting, and B. A. Brice, ibid., 73, 2786 (1951). (4) T. L. McMeekin, M. L. Groves, and N. J. Hipp, Advances in Chemistry Series, No. 44, American Chemical Society, Washington, D. C., 1964, p. 54. (5) J. L. Stoddard and G. S. Adair, J. Biol. Chem., 57,437 (1923). (6) 0. Lamm and A. Poulson, Bwchem. J.,30, 528 (1936). (7) A. Rossi-Fanelli, E. Antonini, and A. Caputo, J. B w l . Chem., 236, 391 (1961). (8) W. H. Orttung, J. Am. Chem. SOC.,87, 924 (1965). (9) Phoenix Precision Instrument Co., Philadelphia 40, Pa. (10) R. Benesch, R. E. Benesch, and G. Macduff, Science, 144, 68 (1964). (11) W. H. Orttung, J. Phys. Chem., 67, 1102 (1963).
NOTES
3189
A, mCc.
550
500
700
650
600
1
t 0.2000
where E(v') is the molar extinction coefficient at V' = l / A ' , M is the corresponding solute molecular weight, P indicates the principal value of the integral, and (An/c)Bpis the contribution of the absorption spectrum to Anlc at V. The spectra of met- and oxyhemoglobin from 250 to 700 mfi13were plotted vs. V. It was assumed that E corresponds to M = 16,700. Each spectrum could be fibted by seven gaussian functions of the type E(V)
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0.1900
t
d
=
c0e- 4
In 2 ( u
- vo)P/y2
the parameters of which are given in Table I. Fitting of the spectra with gaussians is a mathematical step that simplifies the calculations; i e . , if eq. 2 is substituted in eq. 1, the following approximate result may be deduced14
0.1900
where p2 = 4 In 2/y2. For present purposes, eq. 3 is satisfactory for l(v - V O ) / Y ~ 6 2. Far from regions of absorption, when (V - V O ) / Y ~2 2, eq. 1 and 2 become
I
21
20
19
18
17
16
15
14
10-8 v, cm.-'.
Figure 1. Refractive increments for oxyhemoglobin (top) and methemoglobin (bottom) solutions in distilled water at 15". Absorption peaks and half-width are indicated by the horizontal linea below each set of data. The line through each set of points is the sum of a spectral and a residual contribution, the latter being eq. 5. The concentrations, lo%, were aa follows: 0,-4.579, a, -4.636, 0, -10.031 for oxyhemoglobin; 0,-4.537, a, -7.159, 0, -7.747, O-, -10.995, and 0, -14.417 for methemoglobin.
The calibration did not change significantly in cooling the refractometer to 15", the temperature at which data were taken. Values of An/c at 15" are plotted vs. v = 1/X in Figure 1. (An = n - 120, where n and no are the refractive indices of solution and solvent.) The precision of the data is close to the limiting precision of the apparatus.l'
Calculations For present purposes, the Kramers-Kronig relationlg may be put into the form
The contributions of each of the seven gaussians to An/c were then calculated from eq. 3 and 4 and summed to obtain the total contribution for the spectral region 250-700 mp. The calculated contribution was subtracted from the data of Figure 1 and the remainder was plotted vs. v2. A straight line of the form
was fitted to the methemoglobin data by least squares, giving a = 0.1731 f 0.0007 and 10% = 25.3 i 2.4. A straight line with the same slope and a = 0.1753 could be drawn through the end regions of the oxyhemoglobin data. The spectral contributions were then combined with eq. 5 and plotted in Figure 1 for com(12) See, for example, L. D. Landau and E. M. Lifschitz, "Electre dynamics of Continuous Media," Addison-Wesley, Reading, Mass.,
1960.
(13) R. Lemberg and J. W. Legge, "Hematin Compounds and Bile Pigments," Interscience Publishers, Inc., New York, N. Y., 1949, p. 749. The data reported here are for the human (oxy) and ox (met) species, but are in satisfactory agreement with our own Cary 14 messurementson horse hemoglobin in the visible region, aa might be expected from the fact that the spectmm is insensitive to species difference for this molecule. (14) G.E.Pake and E. M. Purcell, Phys. Rev., 74, 1184 (1948).
Volums 69,Number 9 September 1966
NOTES
3190
parison with the data. The agreement is good for methemoglobin, but there are noticeable deviations for oxyhemoglobin that are undoubtedly partly explained by some monochromator slit width averaging of the peaks (2-mm. slits were used and the monochromator dispersion was 6.6 mp/mm.).
Table I : Gaussian Fit of Spectra
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eo
Methemoglobin 630 500 406 388 369 300 265
15870 20000 24650 25800 27100 33300 37750
576 540 450' 416 394 349 2 75
17355 18540 22200 24050 25400 28670 36363
1060 4000 1400 1300 5400 5100 3200
4.2 9.5 125 30 27.0 16.0 31.5
Oxyhemoglobin 550 1100 3200 1400 1200 6200 4220
16.0 15.0 11.0 118 17.0 28.4 35.8
Volume-Energy Relations in Liquids at O O K .
by A. A. Miller General Electric Research Laboratory, Schenectady, N ~ wYork (Received March 16, 1966)
By a special (nonlinear) extrapolation of measured liquid densities, Doolittle' derived the relation, In vo = 10/M, for the specific volumes of n-alkanes in the hypothetical liquid state at 0°K. These values were shown to agree with several earlier, independent estimates.'J Additional confirmation by more recect methods willbe presented later. For ethane, which consists of only two methyl groups, 2ro = 1.396 cc./g. or 21.0 cc./mole of CH3. For the infinite polymethylene chain, uo = 1.00 cc./g. or 14.0 cc./mole of CH2. Comparison of these molar volumes shows that YO(H)= 7.0 cc./mole, with no contribution by the internal carbon atom. Thus, the minimum volume at O'K., where (dE/dv)* = 0, can be attributed entirely to the attraction-repulsion of the peripheral H atoms. By comparison, the van der Waals volumes are u,(CH3) = 13.67, (CHJ = 10.23, (H) = 3.45, and I
(-C-)
= 3.33 c~./mole.~
I
Discussion Earlier result^^^^ gave 104b = 33-36 for other nonheme globular proteins, a significantly higher range than the hemoglobin results reported here. Farultraviolet or infrared transitions of the heme groups would cause greater dispersion in the visible and therefore predict an opposite difference. The peptide chains must therefore be responsible for the difference observed. Variations in amino acid compositionwere considered, but a rough calculation suggested that this effect would not account for the magnitude of the difference. A hypochromic effect in the 19O-mp peptide was also considered (hemoglobin is considerably more a-helical than the other proteins), but the predicted reduction in slope was only a fraction of the observed difference. We have therefore observed a significantly lower dispersion in the globin as compared to other proteins that have been studied, but have not established a quantitative explanation. (15) I. Tinoco, Jr., A. Halpern, and W. T. Simpson in "Polyamino Acids, Polypeptides, and Proteins," M. A. Stahmann, Ed., University of Wisconsin Press, Madison, Wis., 1962,p. 167. (16) K. Rosenheck and P. Doty, Proc. Natl. A d . Sei. U. S.,47. 1775 (1961).
The Journal of Physicd Chemistry
The vaporization energy, BO, for the hypothetical liquid at 0°K. is given by the B constant of the FrostKalkwarf vapor pressure equation: eo = -2.3RB cal./mole.46 Thodos and eo-workers have reported the F-K constants for saturated aliphatic,8 olefinic,7 naphthenic? and aromaticg hydrocarbons, and miscellaneous compounds including CCL.'O It was shown that B is an additive function of the chemical structure. For the n-alkanes,6 following ethane (B = - 1070"),the slope of the linear plot of B us. the number of carbon atoms gives AB = -340"/methylene unit. Hence, BO(CH3) = 2.45 and eo(CH2) = 1.55 kcal./mole, and the ratio is 1.58, which is close to the ratio of the number of H atoms in the two groups. For the eoheaive energy densities, (~/v)o = 117 cal./cc. for CHa ~
~~
-
(1) A. I(.Doolittle, J . Appl. Phys., 22, 1471 (1951). (2) A. P. Mathews, J . Phys. Chem., 20, 554 (1916). (3) A. Bondi, aid., 68, 441 (1964). (4) See E. A. Moelwyn-Hughes, "Physical Chemistry," Pergamon Press, Inc., New York, N.Y., 1961,p. 696 ff. (6) A. A. Miller, J . Phys. Chem., 68, 3900 (1964). (6) N.E.Sondak and G. Thodos, A.1.Ch.E. J., 2,347 (1956). (7) C. H.Smith and G. Thodos, ibid., 6, 569 (1960). (8) G.J. Pasek and G. Thodos, J. Chem. Eng. Data, 7, 21 (1962). (9) D.L.Bond and G . Thodos, &id., 5, 288 (1960). (10) E.C. Reynes and G. Thodos, Ind. Eng. C h m . Fundamatals, 1, 127 (1962).
