thermal perturbation difference spectra and d2o vs h2o isothermal difference spectra of...

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 174, 532-540 (1976)

Thermal Perturbation Difference Spectra and D,O vs H,O Isothermal Difference Spectra of Protein-Model Aromatic Chromophores

JAKE BELL0 AND HELENE R. BELL0

Department of Biophysics, Roswell Park Memorial Institute, Buffalo, New York 14263

Received August 18, 1975

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D,O and H,O spectra of these chromophores. For phenols approximately equal AE values are obtained in both types of spectra, but for their methyl ethers AE values of DrO vs H,O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25-4s D,O or H,O. For phenols in D,O vs H,O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D,O and H,O. The refractive index difference between D,O and H,O was shown to be a minor contribution by means of experiments in which D,O was at 5°C and H,O at 47”C, conditions of equal refractive index (Nan). D,O vs H,O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D,O vs H,O spectra of proteins are discussed.

This report deals with the isothermal difference spectra of phenolic, phenyl, and indolic chromophores in D,O vs H20, with their thermal perturbation spectra, and with the origins of these spectra. Thermal perturbation (TP)’ difference spectra are generated from two portions of the same solution at different temperatures (1, 2). The D,O vs H,O spectra of tyrosyl and tryptophyl chromophores have been used for the study of several proteins (3-5). TP spectra have been used for similar pur- poses (1, 2, 6, 7). Improved understanding of the origins of both types of spectra would be of value. TP spectra appear to arise in part from changes in solvation of the chro-

I Abbreviations used: HBA, 4-hydroxybenzyl alcohol; MBA, 4-methoxybenzyl alcohol; Ac-Tyr- NH,, N-acetyltyrosine amide; Ac-Tyr(O-Me)-NH,, 0-methyl-N-acetyltyrosine amide; RNase, bovine pancreatic ribonuclease (EC 2.7.7.16); Me,SO, di- methylsulfoxide; DMA, N-dimethylacetamide; IT or it, isothermal difference; TP or tp, thermal perturbation.

mophore, presumably from a higher degree of order at lower temperature than at higher temperature (1, 8). On addition of less polar cosolvents (e.g., t-butanol or tet- ramethylurea) but not polar cosolvents (e.g., urea), maxima in Aettp are obtained at the low mole fraction of cosolvent (2) at which maxima or minima are observed in other properties of aqueous solutions (9). These maxima and minima are thought to arise from the induction of a higher degree of order in water.

Some D,O vs H,O spectra and TP spectra of phenols are shown in Figs. 1 and 2; the resemblance is considerable. Kronman and Robbins (10) suggested that the negative extrema at 280-290 nm of D,O vs H,O spectra arise from the lower refractive index of D,O. Positive extrema” are generated by perturbants of higher refractive index than water. But the refractive index

” Unless otherwise noted, references to magni- tudes are to the longest-wavelength extremum.

532

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

THERMAL PERTURBATION AND D,O VS H,O DIFFERENCE SPECTRA

difference between D,O and H20, 0.0047 nDZT’, is small compared with the difference of 0.028 between 20% glycerol (a common perturbant) and H20, although A&s of the difference spectra are of similar magnitude.

MATERIALS AND METHODS

RNase, type RAF, phosphate free, was obtained from Worthington Biochemical Co., Ac-Tyr-NH, from Schwarz/Mann, and D,O, 99.8%) from Bio-Rad Laboratories. D,O was distilled from a small amount of KMnO,, with Kjeldahl traps to prevent passage of spray. A second distillation was stopped after about 75% had been collected. The polymer (Glu,,,,Lys,,Ala,,.jTyr,j),, was obtained from Pilot Chemical Co. Me,SO and N-dimethylacetamide were redistilled under vacuum. Dioxane, spectro- quality, was treated with NaOH pellets, refluxed with and distilled from sodium metal (11). Glycerol (Analar, British Drug Houses), was mixed with 1% of NaBH, and three volumes of H,O or D,O and allowed to stand overnight. After treatment with MB-3 resin, the water was distilled in uocuo. Again, three volumes of H,O or D,O was added and distilled, and the glycerol was distilled at 2-4 mm pressure. Disregarding a possible isotope effect, there should be over 98% exchange.

0-methyltyrosine, from Dr. M. Fleysher of this Institute and from Cycle Chemical Co., contained less than 3% nonmethylated tyrosine, as determined from the spectrum at pH 12. Reagent-grade phenol was resublimed; anisole was redistilled under vacuum.

0-methyl-N-acetyltyrosine amide. Dimethyl sul- fate was added dropwise to a stirred solution of 1 g of Ac-Tyr-NH, in 10 ml of H,O maintained at pH 11 with NaOH. The precipitate was filtered off and recrystallized from isopropanol-methanol. The melting point was 205°C (uncorrected); the content of unmethylated Ac-Tyr-NH, was 2%. The structure is inferred from the method of synthesis and from the neutral and high pH spectra.

4-Methox.ybenz,yl alcohol. One-hundred milliliters of commercial MBA was treated with 5 g of NaBH, for 2 days. The mushy solid was filtered off, and the turbid filtrate was treated with Celite and filtered through a hydrophobic paper. The filtrate was chilled, while vigorously swirled, to crystallize about half. The liquid was removed with a filter stick. The melt from the crystals was distilled at 105°C at 1.5 mm pressure. The high pH spectrum showed no phenolate. The dinitrophenylhydrazine test was negative. The mass spectrum agreed with that published by Pelah et al. (12).

p-H~~droxybenzyl alcohol. Twenty grams of commercial HBA was suspended in 150 ml H,O heated

Ii d a

u 310 X,nm

533

FIG. 1. TP and IT difference spectra of N-acetyltyrosine amide in H,O and D,O. Concentrations of chromophore: 5 x 10m4 M. One volume of 2 M sodium acetate, with sufficient glacial acetic acid to make the pH 5.25, was added to 50 volumes of a 5.1 x 10m4 M chromophore solution. The final sodium acetate concentration was 0.039 M. This procedure was used for the experiments of Figs. 1, 5, 7, 8, and 10. The curved portions of the baselines were places where we were unable to obtain flat baselines. Difference spectra involving deuterated vs protiated solvents were done with a four-cell arrangement. This is ex- plicitly indicated only in Figs. 2 and 3.

near boiling. The gum on the surface was skimmed off. The milky liquid was decanted from the gum on the bottom and was stirred for 1 h with Norit A. Celite was added, and the suspension was filtered. The filtrate was chilled to yield white crystals. The dinitrophenylhydrazine test was negative. The mass spectrum agreed with that of Pelah et al. (12).

Methods. Densities of glycerol-h and glycerol-d were measured at 25.5 ? 0.02”C with a 25-ml pyc- nometer. TP spectra were measured as described earlier (11, and D,O vs H,O spectra by the four-cell method for solvent compensation (13). Refractive indices were measured at 21°C with an Abbe refrac- tometer (calibrated with water).

RESULTS

Refractive Index

We tested the role of refractive index by experiments in which 4-hydroxybenzyl alcohol (HBA) or 4-methoxybenzyl alcohol (MBA) in D,O at 5°C was measured

534 BELL0 AND BELL0

against HBA or MBA, respectively, in H,O at 46-47°C. Under these conditions the refractive indices of the solvents are nearly equal at the Na, line and, if not equal in the absorption range, are more nearly equal than when both solvents are at the same temperature. Figures 2 and 3 show that when the temperature difference is imposed, the difference spectrum is enhanced. For HBA the refractive index equalization spectrum gave AE about 2.7 times Aeit or AetP,27 ys j0cj. If hei, arises solely from refractive index, equalization of the refractive indices should result in a AE equal to AE~,, for the range 47 vs 5°C less Aeit; that is, a Ae about equal to

x, nm

FIG. 2. Thermal perturbation and isothermal D,O vs H,O difference spectra of HBA (6.4 x 10. 4 M).

Chromophores were in 0.039 M ammonium acetate obtained by adding one volume of 2 M ammonium acetate, pH 5.23, to 50 volumes of chromophore solution. This procedure was used for the experiments of Figs. 2, 3,4, 6 and 9. The quadrants shown for two of the spectra represent the contents and temperatures of the four cells; those above the horizontal line are in the “sample” beam and those below in the “refer- ence” beam. For TP spectra at 5 vs 27°C two cells were used. Buffer was present in all cells. For the D,O vs H,O difference spectra at 5 vs 46”C, the chromophore-free H,O and D,O cells should be at 47 and 5°C rather than as shown. The error resulting from the arrangement used is negligible.

L I 11.111, ,I 270 290 310 330 350

FIG. 3. Thermal perturbation and D,O vs H,O difference spectra of MBA (6.4 x 10m4 M). See legend to Fig. 2 for details.

kg 27 vs 5~. If refractive index contributes nothing, the result of the equalization experiment would be Ae = AetP, 47 ys 50c plus A+ or about 2.8 AQ,, 27 ys 50c. The result for HBA is 2.7 times as large as Actp,27 Ls jOc‘ (95% of theory), indicating that the refractive index difference is not important in generating the isothermal D,O vs H,O spectrum. For MBA a similar result is obtained. Similar results were obtained with Ac-Tyr-NH, and phenol.

We also used a visible chromophore, methylene blue, with a spectrum near the Na, line, so that the refractive indices are not very different from those measured at NaD. (The values for H,O are nDz7 1.3323 and ng& 1.3302 (14).) Also, methylene blue has no exchangeable hydrogens. In this experiment the D,O was at 5°C and H,O at 27°C instead of 47°C; the refractive indices are not equal, but the difference is smaller than when both are at 27°C. Figure 4 shows that the D,O vs H,O spectrum does not decrease but grows in magnitude when the temperature difference is established.

Solvent Structure Effects

D,O is thought to be more structured

THERMAL PERTURBATION AND D,O VS H,O DIFFERENCE SPECTRA 535

-. \ / \ / / \ /

/ ’ ’ / I ‘.I 1 SL j- I 575 600 625 Al 615 A0

WAVE LENGTH, “m

FIG. 4. Difference spectra of methylene blue (1.25 x 10.’ M) in D,O vs HzO. Buffer as in Fig. 2.

than is H,O at the same temperature. The experimental.basis for this idea is not clear (15). We accept as true that cold water is more ordered than warm water. If the thermal perturbation spectra arise, largely, from differential ordering of water, we expect isothermal D,O vs H,O spectra. In fact, this is observed (Figs. 1, 5 and 6). The volume difference between 5 and 27°C results in errors varying from 3 to 10%. Since the error is a positive AE, the correction would deepen the negative and diminish the positive extrema, thereby increasing the similarity between the TP and IT spectra.

D,O vs H,O and TP spectra of phenylalanine were measured, because the absence of a hydrogen-donating or -accepting group should bring out the solvent structure effect more clearly than for phenols and ethers, although there will be interactions between the r-electrons and water. Each D,O vs H,O extremum and shoulder has its TP counterpart (Fig. 71, suggesting a relation between the two phenomena. For phenylalanine, Aei, is about 1t5-1/4 AE~,,,

1

FIG. 5. Difference spectra of N-acetyltryptophan amide. 1 and 2, N-acetyltryptophan amide in 50 and 90%, respectively, D,O vs H,O at 27°C; 3, same chromophore in D,O at 5°C vs D,O at 27°C. Concen- tration of N-acetyltryptophan amide: 9.4 x 10-j M.

Buffer as in Fig. 1

t GLU3BLYS24ALA,,TYR,

kl II I ,I ‘I I I I’ I / 260 260 300 320

WAVELENGTH, n m

FIG. 6. Difference spectra of a polymer of the composition shown. Spectrum 1: IT D,O vs H,O at 27°C; spectrum 2: TP spectrum in D,O at 5 vs 27°C. Concentration: 0.46 mgiml. Buffer as in Fig. 2, ex- cept that one volume of stock buffer was added to 25 volumes of chromophore.

suggesting that at 27°C D,O is about 5°C “colder” than HiO, compared with differences in freezing points (3.8”C) and in temperatures of maximum density (7°C). It appears that the structural difference between D,O and H,O at 27°C is much less than that between H,O at 27°C and H,O at 5°C.

(Figure 7 shows the result of a method of correcting for the volume difference between the two temperatures. Two addi- tional H,O cells were used, and to the warm cell was added 0.3% (the density difference) as much phenylalanine as in the main cold cell. The volume error might also be avoided by using temperatures above and below the temperatures of maximum density.)

536 BELL0 AND BELL0

Phenylalanine D,O 27O

X,nm

FIG. 7. TP and IT spectra of phenylalanine in H,O and D,O. Concentration: 0.385 mgiml (2.4 x 10m3 M). There are two TP spectra: (-----I, curve obtained by the usual two-cell method; (-.-,), curve obtained by four-cell method to compensate for the thermal volume change (see text). For clarity the volume-compensated TP spectrum is deleted where it very closely follows the noncompensated spectrum. Buffer as in Fig. 1.

We compared spectra of phenols and their methyl ethers. In Fig. 3 the TP spectrum of MBA in D,O (or in H,O, not shown) is seen to be similar to that of HBA (Fig. 2) while the magnitude of Ae of the D,O vs H,O spectrum of MBA is only 57% that of HBA. Tyrosine and O-methyltyrosine gave a similar result. The methyl group is expected to affect solvation of the oxygen; it has very little effect on AE~,,, or there is a compensating effect.

Figure 8 shows spectra for HBA and

d d a

X,nm

FIG. 8. Difference spectra of HBA and MBA in protiated and deuterated ethylene glycol. Concen- trations of chromophores: 6.4 x 10m4 M. Buffer as in Fig. 1. The glycol concentration is 94%, the remainder being D,O or H,O as appropriate.

MBA in glycol-d vs glycol-h. The spectral differences are marked, indicating an important role for phenolic OD vs OH in glycol solvent. Similar results were obtained with glycerol-d and glycerol-h. These spectra provide evidence for the un- importance of refractive index. Our nDZ1 values for glycerol-h and glycerol-d are 1.4721 and 1.4710, respectively. The exchangeable hydrogens represent 3.26% of the mass of glycerol and 11% of the mass of H,O. The density of D,O is 11% higher than that of H,O. The density of our glycerol-h was 1.2575 g/ml and of our glycerol- d 1.2980, 3.23% higher. The agreement of the experimental and expected densities indicates that the two are of closely similar purity.

D,O us H,O Spectra in Organic Diluents

To distinguish further between the var- ious effects of D,O we measured IT spectra of HBA and MBA in nonprotic solvents containing small amounts of D,O or H,O. The purpose was to obtain the difference spectrum between protiated and deuterated phenol without the complications of the differential effects of bulk solvents. Hydrogen bonding from protiated and deuterated phenol to the organic solvent will differ, with spectroscopic consequences. The results are displayed in Fig. 9 and Table I, together with data for HBA and MBA in D,O vs HzO, glycol-d vs glycol-h, and glycerol-d vs glycerol-h. At low water content we expect that solvent structure will not be significantly different between the solutions containing D,O and H,O. Since the organic solvents are hydrogen acceptors, water will be largely bound to the solvent, especially at the lower water contents, so that the major hydrogen- bonding effect for phenols will be hydrogen donation to the organic solvent. We used solvents of different hydrogen-accepting abilities. For the phenols in N-dimethylacetamide and Me,SO a 16-fold increase in D,O and HzO, from 0.25 to 4%, doubles Aeit/e, indicating that the effect is at or near a plateau. Since the high absorbance of N-dimethylacetamide requires short- path cells and a high chromophore concentration, we used low water content to min- imize solvent structure differences and


higher water content to provide sufficient D,O to insure essentially complete H-D exchange. In the case of dioxane which permits the use of l-cm cells and a low concentration of chromophore, Aeit/E for HBA was the same at 0.75 and 4% D,O and H,O. The Ae, values in Table I may be somewhat low because of dilution of the

s D,O with residual water in the organic a solvent. The agreement between Acit/e for

HBA in N-dimethylacetamide, Me,SO and dioxane at the lower D,O and H,O contents suggests that the error is small, since residual H,O would be especially important at these D,O levels and would not be expected to be the same in all three solvents.

260 280 300 320 x ,nm DISCUSSION

FIG. 9. Isothermal difference spectra of HBA and MBA in organic solvents containing small amounts

In Eq. [l], introduced by McRae (161, Av

of D,O vs H,O. For DMA and Me,SO the optical is the frequency difference between the

thickness is 0.2 mm; concentration of HBA and spectra in the vapor and solution, n and D

MBA, 4.0 x 10m2 M. In dioxane containing 0.75% D,O are the refractive index and dielectric con-

vs 0.75% H,O, optical thickness 1.0 cm, the concen- stant, respectively, A is a dispersion term, tration of HBA is 5.0 x 1O-4 M and of MBA is 5.2 x and B and C are solute dipole moment 10m4 M. Buffer as in Fig. 2. Solid line for HBA, terms. The first refractive ratio and the dashed line for MBA. dielectric ratio are the same to 0.1% for

TABLE I

D/H DIFFERENCE SPECTRAL DATA FOR PHENOLS AND THEIR METHYL ETHERS

Experi- ment

Chromophore pair Solvent pair

1 Tyr:O-Me-Tyr D,O:H,O 0.060 0.026 2 Ac-Tyr-NH,:Ac-Tyr(OMe)-NH, D,O:H,O 0.053 0.031 3 HBA:MBA D,O:H,O 0.053 0.031 4 Phenol:anisole Glycerol-d:glycerol-h 0.032 0.014 5 HBA:MBA Glycol-d:glycol-h 0.023 0.006 6 HBA:MBA Me,SO-0.25% D,O:Me,SO-0.25% H,O” 0.012 0.003 7 HBA:MBA DMA-0.25% D,O:DMA-0.25% H,O’ 0.012 0.003 8 HBA:MBA Dioxane-0.75% D,O:dioxane-0.75% H,O” 0.014 0.002 9 HBA:MBA Me,SO-4% D,O:Me,SO-4% H,O’ 0.021 0.004

10 HBA:MBA DMA-4% D,O:DMA-4% H,O’ 0.022 0.002 11 HBA:MBA Dioxane-4% D,O:dioxane-4% H,O” 0.014 0.002

I1 At for each phenol and ether was obtained from a difference spectrum in deuterated vs protiated solvent. WE is AE,~ divided by E,,, of the direct spectrum in the deuterated solvent. The subscripts OH and OCH, in columns 4 and 5 indicate the phenol and its methyl ether, respectively.

’ Me,SO:water ratio is about 100; water:chromophore ratio is 4. In footnotes b-g the ratios are on a mole basis.

’ DMA:water ratio is about 70; water:chromophore ratio is 4. ” Dioxane:water ratio is 24; water:chromophore ratio is 520. ’ MeSO:water ratio is 6; water:chromophore ratio is 55. ’ DMA:water ratio is 4.5; water:chromophore ratio is 55. * Dioxane:water ratio is 4.5; water:chromophore ratio is 4406.

538 BELL0 AND BELL0

D,O and H,O. The second refractive ratio differs by 1%

b=A+B(~) HI

between D,O and H40, but the value of the whole term differs by 0.25%. These are calculated from n,. Our experimental results are in accord with a minor role for refractive index in D,O vs H,O spectra. The spectra in deuterated vs protiated solvents do not arise from differences in dielectric constants, since the constants are nearly identical for H,O and D,O (17) and are probably nearly identical for deuterated and protiated glycerol. In D,O-5’C vs H,O-47°C experiments we changed the dielectric constants to 86 for D,O at 5°C and 71 for H,O at 47°C (17), changing (D - l)/(D + 2) by 0.6%. This is expected to produce a blue shift (18), increasing the long-wavelength extremum. This is observed. Equa- tion [II, which applies best to non-hydrogen-bonding solvents, concerns the frequency shift only, not the intensity difference which, for small shifts, is propor- tional to Au (18). By cooling D,O and warming H,O we enhanced the structural difference. Tilley (8) proposed that blue shifts for phenols and their ethers in D,O vs H,O or in cold vs warm water arise from greater solvent order.

Estimation of Factors Contributing to D,O us H,O Spectra

1. Phenolic OD us OH. We wish to assign contributions to Aei, from phenolic OD vs OH, hydrogen bonding from D,O and H,O to phenolic oxygen, and solvent structure. Dissection of the effect into these interdependent factors is artificial to some extent but may be illuminating. We esti- mated the contribution from phenolic OD vs OH in three ways. First, comparison of Aeit/e for ethers and phenols (Expt. 1-3, Table I) suggests that half of the phenolic Aei, arises from replacing OH by OD. A second estimate was made in a similar

manner from experiments 4 and 5, to yield values of 25-40% for the OD vs OH contribution. A third method uses the organic- water experiments (Expt. 6-11). Table I shows that he/e for HBA is dependent on the water content. The fact that in experiments 6, 7, 9 and 10, an increase in D,O and Hz0 results in an increase in Ae/e does not mean that bonding of D,O and H,O to HBA is important, since in experiments 6 and 7 the ratio of D,O to chromophore is 4 and HBA has two exchangeable hydrogen atoms. The increase in Ae/e for HBA in experiments 9 and 10 may arise from more complete exchange. This is supported by the observations that in experiments 8 and 11, in which there was sufficient D,O for complete exchange, AE/E is the same for both water contents and that AC/E for MBA is independent of D,O content. Therefore, we suggest that Act/e for phenols in experiments 9-11 arises in large part from replacing OH by OD in the phenols. Since Aeit/e values for the ethers in the organic-water solutions are very small, we assign the whole effect for phenols to replacement of OH by OD. Aeit/E for HBA in dioxane-water (Expt. 8 and 11) is 25% of the average for three phenols in D,O vs H,O (Expt 1-3). From the results from Me&SO and DMA with 4% water, the OD vs OH contribution is 40% of the average of Aeit/e for the phenols in experiments l-3. Thus, the several methods yield OD vs OH contributions of 25- 50% of the total Aeit/e for phenols in D,O vs

H,O. 2. Hydrogen bonding. To estimate

AeitlE for hydrogen bond acceptance in bulk D,O vs HzO, we assume it to be the same for ethers and phenols. Aeit/e for MBA in organic-water solutions is too small because little water is present; Aeit/e for MBA in D,O vs H,O is too large, because structure contributes. For want of some- thing better, we use the average Aeitle of 0.010 for MBA in glycol and for anisole in glycerol as the contribution of the difference between the hydrogen bond acceptance in D,O vs H,O for phenols and ethers. This is about 20% of Aeit/e for phenols in D,O vs H,O (Expt l-3).

3. Water structure. The remaining 30-


55% of Ant/e for phenols in D,O vs H,O is assigned to solvent structure. For phenols Aetr, equals Aei,. The thermal perturbation effect for a temperature difference of 22°C is two to three times as large as the solvent structure contribution to heit. The TP spectra arise from reciprocal hydrogen bonding between phenol and solvent and solvent structure effects. Since Aetp is the same for ethers and phenols, it appears that differences in hydrogen bond donation by phenols to water at the two temperatures used are not of major importance. We shall not attempt to estimate the contributions of these factors.

Relative to spectra in H20, the D,O spectra for HBA and MBA are of reduced intensity, as shown by the nonzero sum of the positive and negative extrema. Bailey et al. (18) attributed intensity changes of phenols in different solvents to differences in hydrogen bonding to solvent. Since MBA cannot donate a hydrogen, yet shows an intensity decrease (smaller than for HBA), the D,O vs H,O spectra arise in part from differences in solvent structure and hydrogen bond acceptance by the chromophore. Since Bailey et al. found almost no change in intensity for phenylalanine in ethanol vs water, they proposed that intensity changes for phenols arise from hydrogen bond donation differences. Our result for phenylalanine suggests that an intensity change may arise from some other effect, such as a difference in solvent structure.

Application to Proteins

Herskovits and Sorensen (4) appear to have obtained valid results with the D,O vs H,O spectrum of pepsin, since with D,O and with organic perturbants they found 50 * 10% exposure of chromophores. For RNase D,O perturbation failed (1) in that apparent exposure of all six tyrosyl side chains was observed instead of the three indicated by other perturbants (1, 13). Other methods demonstrated the excess exposure to be illusory (1, 19). IT spectra at 92% glycerol-d vs glycerol-h gave a value above 6 for the ratio Aeit, RX;r,e/ AEit, mc,dcl. However, the melting profiles (Fig. 10) for RNase in these solvents show

012

I r / 0.10

’ 1 Temperature, OC

FIG. 10. Melting profiles of RNase (7.3 x 10 ’ MI in protiated and deuterated 92% glycerol, containing, respectively, 8% H,O or D,O. Buffer as in Fig. 1. (01, Deuterated (data from two experiments); (0). protiated. On cooling and reheating a T, of 68°C was obtained for RNase in glycerol-h.

that not all tyrosyls are exposed at room temperature. TP spectra of Ac-Tyr-NH, and RNase in 90% glycerol (1) indicated about five exposed tyrosyls, in disagree- ment with chemical reactivity, circular di- chroism and X-ray diffraction, which indicated the persistence of the native confor- mation (20). The high value of exposure may be the result of partial unfolding, since Ae of exposure of one buried tyrosyl chromophore is about 20 times that of Aeit or Aetlr .

Zipp and Kauzmann (21), studying ben- zenoid chromophores in water, noted that, “Without knowing the relative positions and orientations of the water molecules of the cage and the plane of the benzene mol- ecule inside the cage it is not possible to predict whether this interaction should lead to a red shift or a blue shift.” For methyl nitrite, they suggested uncertainty of hydrogen bonding on chromophore-solvent orientation. For models and proteins, D,O produces blue shifts relative to H,O, but neighboring protein groups may affect the structure and orientation of the cage producing larger or smaller blue shifts or even a red shift. Other sources of uncertainty are chromophores largely exposed to solvent, but hydrogen bonded to other groups so that the OD vs OH contribution to AEil is different from that of the model. The X-ray structure of RNase shows several exposed, hydrogen-bonded tyrosyls. The fact that Aetp is the same for phenols

540 BELL0 AND BELL0

and ethers suggests that estimates of exposed chromophores from AQ,, may not be seriously in error from hydrogen-bonded phenols; and in fact, the TP spectrum of RNase in water gives an estimate of exposed tyrosyls in good agreement with other methods (1).

ACKNOWLEDGMENTS

Supported in part by Grant No. GB 20083 from the National Science Foundation, and Grant No. IC- 92 from the American Cancer Society.

REFERENCES

1. BELLO, J. (1969) Biochemistry 8, 4542-4550. 2. PITTZ, E. P., AND BELLO, J. (1970) Trans. Fora-

day Sot. 66, 536-545. 3. DONOVAN, J. W. (1967) Biochemistry 6, 3918-

3927. 4. HERSKOVITS, T. T., AND SORENSEN, M., SR.

(1968) Biochemistry 7, 2533-2544. 5. SOGAMI, M., UYEDA, M., AND OGURA, S. (1973)

Biochim. Biophys. Actu 310, 487-494. 6. BELLO, J. (1970) Biochemistry 9, 3562-3568. 7. LEACH, S. J., AND SMITH, J.A. (1972) Znt. J.

Protein Res. 4, 11-19. 8. TILLEY, G. J. (1967) Thesis, Purdue University,

Lafayette, Ind. 9. FRANKS, F., AND IVES, D. (1966) Quart. Reu.

Chem. Sot. 20, l-44. 10. KRONMAN, M. J., AND ROBBINS, F. M. (1970) in.

Fine Structure of Proteins and Nucleic Acids (Fasman, G. D., and Timasheff, S. N., eds.), p. 282, Marcel Dekker, New York.

11. VOGEL, A. I. (1957) Textbook of Practical Or- ganic Chemistry, p. 177, Wiley-Interscience, New York.

12. PELAH, Z., WILSON, J. M., OHASHI, M., BUDZI- KIEWICZ, H., AND DJERASSI, C. (1963) Tetra- hedron 19, 2233-2244.

13. HERSKOVITS, T. T., AND LASKOWSKI, M., JR. (1962) J. Biol. Chem. 237, 2481-2492.

14. TILTON, L. W., AND TAYLOR, J. K. (1938) J. Res. Nut. Bur. Stand. 20, 419-477.

15. HOLTZER, A., AND EMERSON, F. M. (1969) J. Phys. Chem. 73, 26-33.

16. MCRAE, E. G. (1957) J. Phys. Chem. 61,562-572. 17. EISENBERG, D., AND KAUZMANN, W. (1969)

Structure and Properties of Water, Oxford University Press, New York.

18. BAILEY, J. E., BEAVEN, G. H., CHIGNELL, D. A., AND GRATZER, W. B. (1968) Eur. J. Biochem. 7, 5-14.

19. HERMANS, J., JR., AND SCHERAGA, H. A. (1959) Biochin. Biophys. Actu 36, 534-535.

20. BELLO, J. (1969) Biochemistry 8, 4535-4541. 21. ZIPP, A., AND KAUZMANN, W. (1973) J. Chem.

Phys. 59, 4515-4224.

thermal perturbation difference spectra and d2o vs h2o isothermal difference spectra of...

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