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Interaction of sodium dodecyl sulfate with tyrosyl chromophores in ribonuclease A and model compounds
ARCHIVES
OF
RIOCHEMISTRY
Interaction
AND
RIOPHYSICS
of Sodium
147, 284298
(1971)
Sulfate
with
Dodecyl
in Ribonuclease
A and EUGENE
Department
of Biophysics,
State University
Model
Tyrosyl
Chromophores
Compounds’
P. PITTZ2 of New York at Buffalo,
Bu$alo,
New York
14.!314
AND
JAKE Department
of Biophysics, Received
Roswell
BELL0
Park Memorial
May 5, 1971; accepted
Institute, July
Buffalo,
New York 1,$.203
19, 1971
Ultraviolet difference spectra, solvent perturbation difference spectra, and temperature perturbation difference spectra indicate that tyrosyl residues of model compounds are affected by sodium dodecyl sulfate. This effect is dependent on the nature of the model compound, being enhanced by positive charges, and is attributed to partial masking of the tyrosyl chromophores by sodium dodecyl sulfate. With reduced carboxymethylated ribonuclease as a model, all three difference spectral methods can be interpreted as indicating nearly complete externalization of tyrosyl chromophores in ribonuclease in the detergent. With small tyrosyl model compounds the calculated number of external tyrosyl residues depends on the nature of the model compound. Using net positively charged tyrosyl compounds as models, nearly 6 external tyrosyl residues are calculated for RNase. N-Acetyltyrosine amide or N-acetyltyrosine esters appear to be inadequate models for tyrosine in proteindetergent solutions because of their weak int,eractions with detergents.
Reports of the state of the tyrosyl residues in RNase (polyribonucleotide 2-oligonucleotide-transferase, cyclizing) in the presence of sodium dodecyl sulfate (SDS3) are dis1 This work was supported by grants from the Institute of General Medical Sciences of the National Institutes of Health (GM 13435 to Roswell Park Memorial Institute, and Training Grant GM 00718 to the Department of Biophysics, State University of New York at Buffalo) and from the National Science Foundation (GB 7523 and GB 20083). Th is work is abstracted from a dissertation by E.P.P., State University of New York at Buffalo, 1970. 2 Present address: Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02154. 3 Abbreviations used: RNase, bovine pancreatic ribonuclease, EC 2.7.7.16; SDS, sodium dodecyl sulfate; CD, circular dichroic spectrum; ATA. N-acetyltyrosine amide; RCM-RNase, RNase having its disulfides reduced and carboxymethyl-
cordant. Bigelow and Sonenberg (1) have shown that SDS (> 10 mM) causes a blue shift of the tyrosyl band of RNase with a Ae of -750 indicating exposure of about one additional tyrosyl residue. Herskovits and Laskowski (a), studying the solvent perturbation difference spectrum of RNase in 150 mM SDS, found either no change or a slight decrease in the fraction of accessible tyrosyls depending on the perturbant used. Since there is uncertainty concerning the state of the tyrosyls in RNase in the presence of SDS, we have studied the effect of SDS on the spectral properties of tyrosyl chromophores of RNase and of several model compounds. It appeared important in this type of study to investigate more adequately the effect of SDS on a variety of models varying ated; TP, thermal turbation. 284
perturbation;
SP, solvent
per-
RNase
AND
TYROSYL
in charge and hydrophobic character. Other investigators have omitted SDS from their model systems on the ground that SDS does not affect the chromophores at low SDS concentration. In this communication we present evidence that SDS does affect tyrosyl and tryptophyl chromophores even at rather low SDS concentrations, and that the effect is very strongly dependent on the model compound selected. MATERIALS
AND
METHODS
Materials Ribonuclease A was purchased from Mann Research Laboratories, New York, NY, as were N-acetyl-L-tyrosine amide, gly-tyr-gly, leu-tyrleu, lys-tyr-lys, and glu-tyr-glu. Tyramine hydrochloride was purchased from Calbiochem and Eastman Orga,nic Chemicals. RCM-RNase was prepared by the method of Sela et al. (3,4), except that Cleland’s reagent (dithiothreitol) was used instead of thioglycolic acid. Circular dichroism measurements gave no evidence of a band at 280 nm in RCM-RNaae, indicating loss of native conformation around the tyrosyls. The molar extinction coefficient of this derivative was 8350 at 274.2 rnp using a molecular weight of 14,272. The molar extinction coefficients of RNase and ATA at the 274-278 nm peak were 9650 and 1400, respectively. The coefficients of leu-tyr-leu, lystyr-lys, glu-t,yr-glu, and tyramine were, respectively, 1400, 1320, 1420, and 1450. A value of 1400 was assumed for gly-tyr-gly, but this material contained 1&20y0 of an iodinatable impurity (Dr. 0. A. Roholt, personal communication). The concentrations of these derivatives were estimated from the molar extinction coefficients. Glutathione (oxidized) was purchased from Nutritional Biochemicals Corp., Cleveland, OH. Sodium dodecyl sulfate and hexadecyltrimethylammonium chloride (HTAC) were purchased from Eastman Organic Chemicals and were used without purification. Several experiments were repeated with Sequanal Grade SDS from Pierce Chemical Co., with no difference in results. Urea, sucrose, and sodium acetate were reagent grade. Urea solutions were deionized with MB-3 resin immediately before use. Spectral
Investigations
The temperature perturbation difference spectral method has been developed independently by Bello (5, 6) and Cane (7). Thermal perturbation (TP) difference spectral measurements were carried out as described earlier (5), except that the temperatures used were 27 and 6”.
MODELS
IN
SDS
285
Ultraviolet difference spectra were measured in a Cary 15 spectrophotometer at 27”. The four-cell method of Laskowski et al. (8) was employed to compensate for solvent. For gly-tyr-gly and leutyr-leu the two-cell method was employed, and the difference spectra were corrected for light scattering by the method of Leach and Scheraga (9). Both methods were used for lys-tyr-lys and glu-tyr-glu, the results agreeing within 370. Solvent perturbation (SP) difference spectra were obtained by the method of Herskovits and Laskowski (10). The sample and reference were thermostatted at 27’. There was some difficulty in obtaining flat base lines in a few cases because of very slight differences in SDS concentrations and was easily compensated by correcting for light scattering (9), or by slight adjustments in the SDS concentration in the chromophore-free reference cells. Circular dichroism measurements were taken at 25 and at 4” with a Jasco ORD/5 recording spectropolarimeter with CD accessory. When spectra were taken at 4”, nitrogen gas was used to purge the sample compartment of water vapor. Fluorescence measurements were made at 25” with an Aminco-Bowman spectrophotofluorimeter at an excitation wavelength of 275 nm; the emission was scanned to 500 nm. The slit arrangement, was No. 3. Quantum efficiencies were calculated on the basis of & = 0.014 for ribonuclease A in water at pH 5.8 (11, 12). The fluorescence of SDS was negligible. Enzymatic activity measurements were made by the method of Kunita (13). RESULTS
Ultraviolet Di$erence Spectral Measurements Ultraviolet difference spectral measurements for RNase and RCM-RNase in 30 mM SDS 2)s. RNase and RCM-RNase, respectively, in water are shown in Fig. 1. For solubility, RCM-RNase was studied at pH 2.6 while RNase was studied at’ pH 5.8. No difference spectrum occurs for RNase in 30 mM SDS at pH 5.8 vs. RNase in 30 mM SDS at pH 2.6, indicating that SDS effects on the tyrosyl chromophores in RNase are the same at these pH values. RNase in 30 m&x SDS vs. RNase in water gives a negative difference spectrum similar to that of Bigelow and Sonenberg (1). However, Fig. 1 shows a difference spectrum for RCM-RNase in 30 m&f SDS vs. RCM-RNase in water with a positive AE~ of 1620 at 287 nm, where
286
PITTZ
AND
BELL0
D indicates the classical ultraviolet difference spectrum, as distinguished from the SP and TP difference spectra discussed below. Figure 2 shows positive difference spectra for lys-tyr-lys and tyramine in 150 rnmrSDS vs. these models in aqueous solution. RCMRNase in 150 rnM SDS gives a positive difference spectrum similar to that for RCMRNase in 30 mM SDS. The difference spectral results are summarized in Table I. The AeD values for the tyrosyl model compounds, except for RCMRNase, are 6 times the AcI, measured, for comparison with RNase and RCM-RNase, which contain 6 tyrosyl residues. AeD is negaF’ tive for RNase at all SDS concentrations. RCM-RNase, ATA, gly-tyr-gly, leu-tyr-leu, A jrnp lys-tyr-lys, and tyramine give positive Fro. 2. The uv difference spectra of model comAE~ values. AQ for glu-tyr-glu, which has a pounds in 150 mM SDS 8s. these solutes in water. net negative charge at pH 5.8, is effectively 1, lys-tyr-lys (6.35 X lo-+ M), pH 5.8; 2, tyramine zero in 30 and 150 rn>r SDS. (1.1 X 10e3 M), pH 5.8; 3, RCM-RNase (2.82 X We shall assume here that SDS enfolds lo-* M or 1.7 X lo-” M in tyrosyl residues), pH 2.6. the tyrosyl chromophore, part,ially masking Each unit on the AOD scale represents 0.05 OD. it from other components of the solvent. The O-l slidewire was used. There is the alternate possibility that SDS does not enfold the tyrosyls, but causes them This is not possible for tyramine but is posto be buried within the protein or peptide. sible for protein and partially so for the tripeptides. We shall consider the folding hypothesis in the Discussion; here we shall lsee the conclusions reached on the hypothesis of masking by SDS. We shall use the definif tions: buried, enfolded within the protein; masked, shielded by SDS from other solvent components; exposed, outside the protein and not masked; external, outside the protein, whether masked or not. By subtracting AQ, for RCM-RNase, or 6 AQ, for small models, from Ae, for RNase we compensate for the masking of the tyrosyl by SDS. This is the same type of correction that has been made for other denaturants such as urea and LiBr (14, 15). Column 4 of Table I shows these corrected AeD values. Fully denatured RNase has a AE~ of about -2700 relative to native RNase (14). If this -I represents exposure of all of the buried FIG. 1. The uv difference spectra of RNase and tyrosyls upon denaturation, we can calcuRCM-RNase in 30 mu SDS vs. these solutes in late the number of external tyrosyls in water; (a), RNase (7.7 X 10e5 M) in 30 mM SDS vs. RNase in SDS solution (Table I). The calRNase in water, pH 5.8; (b), RCM-RNase (6.1 X culation depends on the chosen model. For W5 M) in 30 mM SDS vs. RCM-RNase in water, lys-tyr-lys in 30 rnbr SDS Ae, is about the pH 2.6. Each unit on the AOD scale represents 0.01 same as in 150 ml\r SDS. For ATA Ae, is unit OD.
RNase AND TYROSYL
MODELS
TABLE THE uv DIFFERENCE
IN SDS
I
SPECTRAL RESULTS FOR RNASE, RCM-RNASE, vs. THESE SOLUTES IN WATER
Solute”
RNase, 10 mM SDS RCM-RNase, 10 mM SDS ATA, 10 mM SDS RNase, 20 mM SDS RCM-RNase, 20 mM SDS ATA, 20 mM SDS RNase, 30 mM SDS RCM-RNase, 30 mM SDS ATA, 30 mrvr SDS Gly-tyr-gly, 30 mM SDS Leu-tyr-leu, 30 mM SDS Glu-tyr-glu, 30 mu SDS Lys-tyr-lys, 30 mM SDS RNase, 52.5 rnx SDS RCM-RNase, 52.5 mM SDS ATA, 52.5 mM SDS RNase, 150 mru SDS RCM-RNase, 150 mM SDS Lys-tyr-lys, 150 mM SDS Tyramine, 150 mM SDS Glu-tyr-glu, 150 mM SDS
Aebsc D
-920 +1450
+6’3
(+lO)
-860 +1570
+120
287
(+20)
- 1010 +1620 +294 (+49)
+216 (+36) +492 (+82) -36 (-6) +1632 (f272) -980 +1570
+480 (+80) - 1010 +1840 +1674 (f275) +1790 (+298)
+36 (+6)
AND MODEL PEPTIDES
AD b-d
AED (corrected)a
286.9 285.5 284.7 286.3 285.6 284.7 286.2 285.8 284.8 284.0 285.1 284.0 284.6 286.8 285.8 284.6 286.5 285.5 284.7 284.5 284.0
-2370 -980 - 2430 -980 - 2630 - 1304 - 1226 - 1502 -974 - 2724 - 2550 - 1460 - 2850 -2684 -2800 - 1046
IN
SDS
5.7 4.3 5.7 4.2 5.9 4.6 4.5 4.8 4.3 6.0 5.9 4.9 6.1 6.0 6.1 4.4
= RCM-RNase is at pH 2.6. All other solutes are in 0.01 M sodium acetate buffer, pH 5.8. b The AND listed here for ATA, gly-tyr-gly, leu-tyr-leu, lys-tyr-lys, and glu-tyr-glu and tyramine are 6 times the Aeo actually measured. This enables us to compare ANDfor RNase with AEDfor the model compound, since RNase contains six tyrosyl molecules. The number in parentheses is the observed A6n . c The difference spectra of gly-tyr-gly and leu-tyr-leu are corrected for light scattering by the method described by Leach and Scheraga (9). This was necessary since only two cells were used to obtain difference spectra,. d The difference spectral data are not corrected for the effect of SDS on disulfide absorbance since a AED of only 2 was found for oxidized glutathione in 30 mu SDS vs. glutathione in water. Even if glutathione is not a perfect model the error must be negligible. c The number of exposed tyrosyl residues listed is based upon the fact that a AEDof -2700 represents the exuosure of all of the buried tvrosvl residues of RNsse. A~D (corrected) is ANDfor RNase minus Ac, for the model for which the value is shown.
small in 10 mM SDS and increases with increasing SDS concentration. Ae, values for RNase and RCM-RNase in 10 rnbr SDS reach 90 and SO%, respectively, of AED in 150 mM SDS, indicating that the effect of SDS on the tyrosyl chromophores of these proteins is nearly complete at 10 mM SDS. (Possibly Ace would be still larger and masking more complete at much higher SDS concentrations.) We have also measured difference spectra for RNase, glu-tyr-glu and tyramine, at pH 5.8, in 30 m&r hexadecyltrimethylam-
monium chloride vs. these solutes in water. Glu-tyr-glu gives AcD of +500 (3000 for 6 AED) at 286 nm while RNase and tyramine had AcD of +30 and +60, respectively. The CD spectra of RNase in water and in this detergent were the same at 240-300 nm. Solvent Perturbation Dierence Spectra The SP difference spectra for RNase and RCM-RNase in water and 150 mM SDS with sucrose as perturbant are shown in Fig. 3. SDS (150 mM> depresses the SP spectrum of RNase, and even more so, that
288
PITTZ
AND BELL0
of RCM-RNase. Figure 3c shows the depression of the SP spectrum of lys-tyr-lys in 150 mM SDS as compared to this model compound in water.
kmr FIG. 3. Solvent perturbation
A, w
difference spectra in water and 150 lll~ SDS. a: 1, RNase (2.4 X lo-’ M) in water, pH 5.8; 2, RNase (2.4 X lo-* M), in 150 m SDS, pH 5.8; b: 1, RCM-RNase (2.8 X low4 M), in water, pH 2.6; 2, RCM-RNase (2.8 X 1V4 M), in 150 lll~ SDS, pH 2.6; c: lys-tyr-lys (1.2 X 10-’ M); 1, water, pH 5.8; 2, in 150 mM SDS. For RNase and RCM-RNase 20% sucrose is used as perturbant. For lys-tyr-lys, 10% sucrose is used as perturbant. Each unit on the AOD scale represents 0.01 unit OD.
Figure 4a indicates that Acsp/ena vs. sucrose concentration is linear for RNase and model compounds in aqueous solution up to 20% sucrose, and that the slope for RNase is about 0.55 t’hat of the others, indicating the fractional exposure of tyrosyls. In 150 mM SDS (Fig. 4) only glu-tyr-glu shows linear behavior, with slope identical to that in wat’er, while RNase and the other models show nonlinearity, the deviation being greater for lys-tyr-lys and tyramine than for RNase and RCM-RNase. The deviation is reproducible. Thus the calculated exposure of tyrosyls depends not only on the model compound but also on the concentration of perturbant, especially in multicomponent systems. The significance of the nonlinearity will be considered in the Discussion. Table II summarizes the results of the SP difference spectral method. Column 5 shows the fraction of external tyrosyl residues calculated by dividing A+,/E~ for RNase by AGJQ for the model compound at the same SDS and perturbant concentration. Column 6 gives the fraction of external tyrosyl residues of RNase multiplied by 6, to give a quantity which we propose is equal to the number of external tyrosyl residues of RNase. 2.9-3.5 tyrosyl residues are found to be exposed (= external in this case) for RNase in aqueous solution. For RNase in 150 m&T SDS the range of values is 4.4-6 external tyrosyl residues, except with glu-
FIG. 4. Aesp/c~ vs. sucrose concentration. (a), in water; (b), in 150 rnM SDS. 0, glu-tyr-glu; n, tyramine; 0, lys-tyr-lys; 0, RNase; & RCM-RNase. The horizontal scale is offset to accommodate all the plots.
RNase
AND
TYROSYL TABLE
SUMMARY
MODELS
289
SDS
II
OF SOLVENT PERTURBATION DIFFERENCE SPECTRAL RESULTS FOR RNASE COMPOUNDS IN WATER AND 150 mM SDS USING 10 AND 20% BY WEIGHT SUCROSE AS PERTURBANT % Sucrose perturbant
system
RNase,
IN
Fraction of external tyrosinesC
AND MODEL
Fraction X6O
Hz0
0.010
236.0
10
-
-
RCM-RNaae, H20 Lys-tyr-lys, Hz0 Tyramine, Hz0 Glu-tyr-glu, Hz0 RNase, 150 mM SDS
0.018 0.020 0.021 0.021 0.006
284.5 284.5 284.0 284.5 285.5
10 10 10 10 10
0.55 0.50 0.48 0.48 -
3.3 3.0 2.9 2.9 -
RCM-RNase, 150 mM SDS Lys-tyr-lys, 150 mm SDS Tyramine, 150 mu SDS Glu-tyr-glu, 150 mu SDS RNase, Hz0
0.007 0.006 0.007 0.021 0.021
284.5 236.0 236.0 234.5 286.0
10 10 10 10 20
0.9 1 0.9 0.3 -
5 6 5 2
RCM-RNase, Hz0 Lys-tyr-lys, Hz0 Tyramine, Hz0 Glu-tyr-glu, Hz0 RNase, 150 mM SDS
0.036 0.038 0.041 0.039 0.016
284.5 284.5 284.0 284.0 285.0
20 20 20 20 20
0.58 0.55 0.51 0.54 -
3.5 3.3 3.1 3.2 -
RCM-RNase, 150 mra SDS Lys-tyr-lys, 150 mM SDS Tyramine, 150 mM SDS Glu-tyr-glu, 150 mM SDS
0.016 0.020” 0.022 0.039”
284.5 286.0 285.0 284.5
20 20 20 20
1.0 0.80 0.73 0.41
6.0 4.8 4.4 2.5
a eM is always measured in aqueous solution without SDS or perturbant. h x,, is rounded to the nearest 0.5 rnr. c Each value shown is for RNase, using as model the compound for which
tyr-glu as model. For glu-tyr-glu the calculated number of external tyrosyls is similar to the number of exposed tyrosyls calculated by Herskovits and Laskowski (2). This is to be expected since these workers did not use SDS in their model system and glu-tyr-glu appears not to interact with SDS. With 20% sucrose as perturbant A~,,/Q for RNase in water is 0.021, in agreement with the value of 0.020 of Herskovits and Laskowski (2). For RCM-RNase we found 0.036 compared with 0.043 of these workers. In 150 mM SDS we found 0.016 for RNase compared with 0.020 of Herskovits and Laskowski (2). Temperature Perturbation Difference Spectral Results The TP difference spectra of RNase and RCM-RNase in the presence and absence of 30 mM SDS are shown in Fig. 5. From the negative extremum (at 286-290 nm) in each of the curves Ae,, is calculated, and the
-
the value is given.
ratio of Actp for RNase to Aetp for the model is used to estimate the external t,yrosyl residues. Figure 6 shows that Aetp for RNase in 30 mM SDS and RCM-RNase in water and in 30 mM SDS is linear with temperature, indicating the probability of no significant conformational transition affecting Actp over this temperature interval. Earlier (5) it was shown that Ae,, of RNase in water is linear from 4 to 25”. Additional support for the absence of a major conformational change is that exposure or burial of a single tyrosyl produces a AE about 3-6 times as great (depending on wavelength) as Ae,, for the entire RNase molecule. This would dominate the spectrum, giving essentially a denaturation or renaturation spectrum (6). Table III summarizes the TP spectral data. The Act* values are not corrected for the small (5) contribution from buried or masked chromophores arising from the volume change on cooling. The decreasing Act, for ATA with increasing SDS concen-
PITT2
AND BELL0
compound, and 3.5 when ATA is the model. For RNase in 8 M urea, 5.6 tyrosyl residues are found exposed; this probably represents real burial because in urea the number of exposed tyrosyls is pH dependent, becoming 5.9-6.1 at pH 4-5. Ae,, of RNase is constant in O-53 mM SDS; Aetp of RCM-RNase changes on transfer in O-10 mM SDS and remains constant in lo-53 mM SDS; Act,, for ATA does not change on transfer in O-10 mM SDS but changes continuously (with decreasing slope) in lo-53 mM SDS. FIQ. 5. The TP difference spectra. Spectrum 1, RNase (7.3 X 10-6 M) in 0.01 M sodium acetate buffer, pH 5.8; 2, RNase (7.7 X 1OU5M) in 30 mu SDS, 0.01~ sodium acetate buffer, pH 5.8; 3, RCMRNase (6.1 X low6 M), pH 2.6; 4, RCM-RNase (6.1 X lO-&M) in 30 nm SDS, pH 2.6. Each unit on the AOD axis represents 0.01 unit of OD. Zero AOD for each spectrum is the horizontal long wavelength region.
tJ 0
0
6
IO
Fluorescence Studies Relative fluorescence emission values, Q/&o, were calculated for RNase and RCM-RNase in SDS relative to water. The data in Table IV indicate that the quantum efficiencies of both RNase and RCM-RNase increase on transfer from water to SDS solutions. The RFE values for RNase and RCM-RNase are almost constant in 2-53 mM SDS indicating that the entire effect is observed at the lower detergent concentration. Enzymatic Activity RNase in 8 M urea was 80% as active as in water. It is known that RNase in urea refolds in the presence of substrates (16). When 30 m&l SDS was added to RNase in water or in 8 M urea there was no spectral evidence of enzymatic activity even after 24 hr of incubation with substrate.
14
TEMP.
16
22
26
*C
FIG. 6. Act, as. temperature. 0, RNase in 30 rnM SDS; n , RCM-RNase in water; and 0, RCMRNase in 30 mM SDS. The base line was set at 27”.
tration indicates that Ae,, for masked tyrosyls is smaller than for exposed tyrosyls. Column 5 of Table III lists ratios of Actl, for RNase to A+, for RCM-RNase and for ATA in the same solvent system. Provisionally we assume that only chromophores exposed to water give Aetp , and that this ratio represents the fraction of external tyrosyl residues in RNase. This will be considered in the Discussion. For RNase in water, 3.4 t)yrosyl residues are exposed to solvent when RCM-RNase is the model
Circular Dichroism (CD) and Transition Temperature Measurements For RNase in water and 20% sucrose identical spectra were found between 210 and 310 nm. In the 240-310-nm region the spectra were similar to those found by Simmons and Glazer (17) both in water and in 20% sucrose. EL - ER at 280 nm was reduced by 87 % for RNase in 150 mM SDS (in agreement with Simmons and Glazer). For both RNase and RCM-RNase in SDS the CD spectrum in the 210-220-nm region indicated an increase in helical and/or /3structure in agreement with the findings of Jirgensons and Capetillo (18). In regard to the TP spectra, the CD spectrum of RCM-RNase in water and 20 % sucrose was
RNase AND TYROSYL TABLE
MODELS
IN SDS
291
III
SUMMARY OF TP DIFFERENCE SPECTRAL RESULTS ON RNASE, RCM-RNASE ATA IN WATER, 8 M UREA AND SDS
AND
Solute RNase, water RCM-RNase, water ATA, water RNaae, 8 M urea RCM-RNase, 8 M urea ATA, 8 M urea RNase, 10 mu SDS RCM-RNaae, 10 mM SDS ATA, 10 mM SDS RNase, 20 mu SDS RCM-RNaae, 20 mM SDS ATA, 20 mM SDS RNase, 30 mu SDS RCM-RNase, 30 mM SDS ATA, 30 mM SDS RNase, 52.5 mM SDS RCM-RNaae, 52.5 mu SDS ATA, 52.5 mnd SDS
-198 -328 -318 -308 -320 -312 - 198 -181 -318 - 194 -184 -246 - 199 - 180 -228 -196 -181 -210
(-53) (-52) (-53)
(-41) (-38) (-35)
-184 -328 -318 -296 -320 -312 - 185 -181 -318 - 185 -181 -246 - 185 -181 -228 -185 -181 -210
289.5 286.3 286.2 288.0 287.4 287.0 289.0 288.9 286.2 289.2 289.5 286.5 289.1 2239.4 286.9 289.1 289.3 287.0
0.56 0.58 0.93 0.94 1.02 0.58 1.02 0.75 1.02 0.81 1.02 0.88
3.4 3.5 5.6 5.6d 6.1 3.5 6.1 4.6 6.1 4.9 6.1 5.3
0 For ATA AE$,,not in parentheses is 6 times the difference in molar extinction shown in parentheses. 5 Act,, (corr) takes into account the Aetp for the four disulfides in RNase using oxidized glutathione in water (Aetp = 3.8) and in SDS (Aetp = 3.2) aa the model compound. c Number of external tyrosyls in RNase, calculated from &Q for the model indicated. d At pH 5.5, 6.0 exposed tyrosines were found, corrected for disulfides. TABLE
IV
SUMMARY OF FLUORESCENCE EMISSION RESULTS FOR RNASE AND RCM-RNASE IN WATER AND SDS
the same at 25 (with or without at 4 than at 25” was no difference syl region.
and 4“. The CD in SDS sucrose) was 10% greater at 210-215 nm, but there above 232 nm in the tyro-
Solute and solvent
DISCUSSION RNase, RNase, RNase, RNase, RNase, RNase,
water 2 mM SDS 10 rnM SDS 20 mM SDS 30 mM SDS 52.5 mM SDS
RCM-RNase, RCM-RNase, RCM-RNase, RCM-RNase, RCM-RNase, RCM-RNase,
water 8 M urea 1.0mM SDS 20 mM SDS 30 mM SDS 52.5 mM SDS
0.014 0.034 0.036 0.036 0.036 0.036
1.0 2.4 2.6 2.6 2.6 2.6
0.035 0.040 0.056 0.055 0.057 0.057
1.0 1.1 1.6 1.6 1.6 1.6
a All measurements were done at a protein concentration of 1.3 x 10e6 M.
Before considering the results we shall recall several possible states of tyrosyl residues : (1) buried inside the protein in native or nonnative conformation; (2) exposed to solvent without masking; (3) externalized and masked by SDS. The idea of masking of chromophores by detergent has been considered by several investigators [e.g., Refs. (2), (19), (ZO)], some of whom have not considered the possibility very likely. Reynolds et al. (21) studied the spectral effect of low levels of alkanoates on serum albumin and proposed the red shift to be “possibly induced by the close proximity of the hydrocarbon tail to the aromatic
292
PITTZ
AND BELL0
residues.” We agree and suggest that such an effect can be hidden in a net blue shift. Cowgill (11, 12) showed that the tyrosyl fluorescence of RNase increases in the presence of SDS, reaching a plateau at 3-10 mM SDS; Table IV shows that there is no further change up to 53 mM SDS. Our data are similar to Cowgill’s and extend to the SDS concentrations used in our other experiments. Cowgill (22) showed that reduction of the disulfide cross-links with formation of new, non-cross-linking disuljides (by oxidizing reduced RNase in the presence of a mercaptan) resulted in quantum efficiencies in 8 no urea or 5 mM SDS the same as for unreduced RNase in these solvents. He also showed that RCM-RNase gave larger quantum efficiencies than did RNase or the derivative just described. Cowgill’s results indicate that disulfides quench tyrosyl fluorescence, and that the high quantum efficiency of RCM-RNase in SDS does not necessarily mean that in SDS the tyrosyls of RCM-RNase are in much different environments than in RNase. Cowgill (22-24) showed that the peptide groups, disulfides, and carboxyl groups can quench tyrosyl fluorescence. If SDS causes burial of tyrosines we do not know whether or not to expect an increase in fluorescence because of the presence of internal quenchers. Externalization and masking of tyrosyls inside the nonpolar portion of SDS is consistent with the decreased quenching. However, titration results (26) indicate an interaction between the tyrosyl hydroxyl and the charged end of the detergent. Circular Dichroism There have been several investigations of the ORD and CD properties of RNase in SDS (17, 18, 27, 28). ORD and CD investigations in the far ultraviolet, which are related to the secondary structure, are not directly relevant to the tyrosyls. The rotatory properties at the 280 nm tyrosyl extremum are of more direct interest. Simpson and Vallee (27) attribute the 280 nm CD band to buried tyrosines. Simmons and Glazer proposed that the 280 nm CD band arises from exposed tyrosines. Pflumm and Beychok (29) and Horwitz et al. (30) consider exposed and buried tyrosyls, as well as
disulfides, to be involved. Horwitz et al. proposed that a significant portion of the CD extremum arises from the most buried tyrosyl. The nearly complete disappearance of the 280 nm extremum suggests that the conformation of RNase in SDS is so disrupted as to cause a gross change in the environment in this tyrosine. Burial in nonnative conformation is not irreconcilable with the CD data. We might expect that such buried tyrosyls would be in environments that would give rise to a CD band; this is not conclusive. State 2, exposed but not masked, is consistent with the CD data and is awkward, but not impossible, to reconcile with the AC, data, which appear to indicate that only one more tyrosyl becomes exposed; that is, the CD extremum might vanish as the result of the sum of small changes among several tyrosyls. The CD result is easily rationalized for State 3, tyrosyls externalized and masked. We propose, then, that the elimination of the 280 nm extremum of RNase by SDS can be explained by disruption of the conformation, and externalization of the tyrosyl residues. This is consistent with fluorescence and AeD . Ultraviolet Difference Spectra The fundamental question is that of the appropriateness of the model system, both as to solvent and chromophore. Bigelow and Sonenberg (1) did not compensate for the effect of SDS on model chromophores; they indicated that Yanari and Bovey (32) found only a small red shift in the spectrum of N-acetyltyrosine ethyl ester in SDS above the critical micelle concentration. Actually, AC, for acetyltyrosine ethyl ester in the 225 rniM SDS used by Yanari and Bovey was +250 at 287 nm (+ 1500 for 6 tyrosyls). While Ae, is small for ATA at low SDS concentration, such as 10 mM, it is much larger for some other models. Should the corrections shown in Table I be made? If the tyrosyls of RNase are buried and not masked by SDS, or if some are exposed and the effect of SDS is solvent perturbation similar to that of sucrose, not arising from a strong association, the correction is small or inappropriate. The data of Table I show a significant effect of SDS on some models,
RNase
AND
TYROSYL
This cannot be a simple solvent perturbation as it is as much as 100 times that of sucrose, normalized for concentration. So large an effect means a strong association, i.e., masking. For RNase, RCM-RNase, and the tripeptide models, it is conceivable that the AeD arises from folding of the molecules to bury tyrosyls. This is not possible for tyramine, which is too small for any significant degree of folding. Yet tyramine shows a A+ as large as any other. Since there is binding of SDS Do the chromophore of tyramine, it is likely that it also occurs with RNase. Therefore, we must make a correction in Ae, of RNase for the masking. The observed AQ, for RNase in SDS might arise from several combinations of exposure, masking, and burial. This will be considered further in connection with Aesp and AQ,, . In Table I using the corrections for RCM-RNase and for the positively charged models we have calculated that all of the tyrosines are external and masked. Perhaps the correction should be less than 6 AQ, for lys-tyr-lys or tyramine because some tyrosyl regions of RNase lack positive charges. But the titration data on aminoacet,ylated RNase (26) show that for the protein, in contrast to the tripeptides, positive charges are not important. The nearly zero AC, for RNase in 30 mM hexadecyltrimethylammonium chloride might have been interpreted as indicating either no effect, or complete externalization and masking of tyrosyls, i.e., about - 2700 for exposure counterbalanced by about +3000 (6 times AE~ for glu-tyrglu) for masking. But the normal CD spectrum at 260-300 nm indicates that the cationic detergent does not disrupt the conformation. In the case of SDS the disappearance of the tyrosyl CD extremum supports the interpretation of disruption of conformation with externalization and masking of tyrosyls. AQ, values for lys-tyr-lys, tyramine, and RCM-RNase are about one-third of Ae for exposure of one buried tyrosyl of RNase (14). This suggests that masking is only partial. HTAC gives a AE~ with glu-tyr-glu almost twice as large as does SDS with tyramine. If this represents more complete
MODELS
IN
SDS
293
masking, perhaps SDS can mask some tyrosyls of RNase or RCM-RNase more completely than it masks tyramine. If so, the Ae, of +1620 for 0.03 M SDS with RCMRNase might agree with 6 AQ, for tyramine because some tyrosyls of RCM-RNase have greater and some have lesser AQ, values. But the long wavelength absorption maxima of lys-tyr-lys in 30 mM SDS and of glu-tyrglu in 30 mM HTAC are the same (26). Also A+, is about only 10% greater for RCMRNase in 150 mM SDS than in 30 mM. If HTAC masks more completely it may have a greater affinity; if so, why does it seem t’o be a weaker denaturant? Reynolds and Tanford (33) have presented data which suggest that below 5 mM SDS, monomer, not micelle, binds to protein. That the effect of SDS on RNase and RCMRNase appears to be largely complete at the lower concentrations of SDS may arise from the binding being largely dependent on SDS monomer concentration, which is constant over the range studied. However, binding to ATA is dependent on total SDS concentration and, presumably, on SDS micelles. Binding of SDS to protein (at high enough concentration to cause disruption) is regarded as largely hydrophobic. Binding to small models is highly dependent on charge. This does not necessarily mean that the small models are invalid. Reynolds and Tanford (34) found that non-disulfide proteins in SDS appear to be rods of similar radius and of length proportional to molecular weight and quite unlike the proteins in the absence of SDS. If the disulfides are intact the elution position (gel chromatography) of the protein-SDS complex is different from that of the complex wit’h reduced protein (35). Reduced proteins bind about 1.4 g SDS/g protein, and unreduced proteins about 1 g/g (33, 36). Even though binding of SDS to unreduced protein is less than to reduced protein, it is highly likely t’hat the conformation of the unreduced protein is disrupted. For unreduced bovine serum albumin Fish et al. (35) found that the Stokes radius of the SDS complex was similar to that of the protein in 6 M guanidinium chloride. This suggests gross disorganization.
294
PITTZ
AND BELL0
Solvent Perturbation Di$erence Spectra Herskovits and Laskowski (2) in studying solvent perturbation difference spectrophotometry of RNase in 150 mM SDS did not use SDS in the model system. They found A’sP/e~ to be 0.020 for RNase in 150 mM SDS using 20 % sucrose as perturbant, while we find Acsp/cM to be 0.016. (0.029 given by Herskovits and Laskowski for the perturbant sucrose in their Table III should be 0.020; T. T. Herskovits, personal communication.) They suggested that the exposure of tyrosyls of RNase in water and in 150 mM SDS is the same, about 50 %. An alternate explanation proposed by them was that SDS binds to tyrosyls, masking them from the perturbant, although no test was done. If there is no masking of tyrosyls by SDS, the conclusion of Herskovits and Laskowski that exposure is about 50% (44% from our data) in 150 mM SDS would probably be correct. If there is masking of tyrosyls by SDS, the procedure of Herskovits and Laskowski can provide no information on the number of external tyrosyls. On the hypothesis that tyrosyls of RNase and of appropriate models are partially masked, we calculate a variety of estimates of external tyrosyls for RNase in 150 m&r SDS. Glu-tyr-glu, which probably is not masked, gives the lowest estimate of external tyrosyls, similar to that of Herskovits and Laskowski based on acetyltyrosine ethyl ester without SDS. RCMRNase, which is expected to be most like RNase gives 5-6 external tyrosyls at lo-20% sucrose. From Table II we see that the SDS-model interactions decrease Aesp/eM by about 50%. Therefore, if 50 % of the tyrosyls _.^. are external, as in native RNase, and if they interact with SDS as do those of the positively charged models, the observed Acs,/cM (with 20% sucrose) would be about 0.01, not 0.02, indicating 50 % external tyrosyls with our model system, and 25% exposed tyrosyls with the model (no SDS) of Herskovits and Laskowski. The crucial point lies in the terms external and exposed. If all of the tyrosyls are external, but are, on the average, 50% exposed (i.e., 50% masked), we have concordance between the interpretations of Herskovits and Laskowski and of ourselves.
The linearity of the Hamaguchi-Kurono plots (37) (Fig. 4a) for RNase and the models in water indicates that access of the perturbant to the chromophore is independent of perturbant concentration. The upward deviation of AEJE~ for SDS and 20% sucrose does not necessarily indicate that 20 % sucrose causes some dissociation of SDS from the tyrosyl to give a AEJE~ close to that in the absence of SDS. Dissociation would decrease AEJQ because the shift of tyrosyl from hydrophobic medium to water would cause a blue shift greater than the red shift of solvent perturbation, unless there were a red shift arising from burial. The increased AQ,/Q at 20 % sucrose may mean that with increasing sucrose concentration there is relatively increased binding of sucrose and/or SDS. Gratzer and Beaven (38) found that sucrose slightly decreased, and ethylene glycol increased the critical micelle concentration of a nonionic detergent. A more dramatic effect of a cosolvent has been observed by us when 20 % ethanol or 2-methyl-2,4-pentanediol is added to RNase in 30 mM SDS: the 280-nm CD extremum is restored to that of native RNase. (This supports the idea that charges are less important than hydrophobic forces in the binding of SDS to RNase.) In the case of the solvent perturbation difference spectral results, we again face the problem of the appropriateness of the models, especially of RCM-RNase. In water RCMRNase appears to be well exposed to solvent since the solvent perturbation spectra of Herskovits and Laskowski (2, 39) and of ourselves indicate similar Aesp/eM for RCMRNase and for ATA. In water RCM-RNase is well unfolded (4042). That a residuum of regular structure remains is suggested by CD studies of Klee (43) on S-peptide. It is encouraging to note that two small models give results similar to those of RCM-RNase, and that AeD for ATA approaches that of RCM-RNase as the SDS concentration increases. In SDS, our conclusions are valid only if the tyrosyls of RCM-RNase and RNase are in substantially similar states. Two of the tyrosyls of RNase are adjacent to d&sulfides, a third is two residues away, and a fourth is three residues away. This situation does not exist in RCM-RNase
RNase AND TYROSYL MODELS IN SDS Titration data (26) suggest that the tyrosyls of RCM-RNase interact somewhat differently with SDS than do those of RNase; but this is a high pH result and may not apply at the pH of this work. We cannot be perfectly confident about the appropriateness of RCM-RNase as a model; but no model can be exactly like RNase. With lys-t,yr-lys and tyramine, we calculate smaller fractions of external tyrosyls in RNase from larger AesP/ean values. The far ultraviolet CD data show that SDS induces an apparently more regular conformation in RCM-RNase. Even if SDS promotes some increase in helical content, the effect on the tyrosines is unknown, and helix formation need not bury tyrosyls. Tyrosyls in an SDS-helical region might be external and masked by SDS rather than buried in the protein. Perhaps SDS acts as “glue” between hydrophobic residues. terhaps tyrosyls are both buried and masked in a proteinSDS complex. The normally helical regions of RNase do not contain tyrosine, and the sequences in which tyrosines are found are not conducive to helix formation. Tyr-25 is in the sequence ser-ser-ser-asn-tyr-cys; tyr-73 and tyr-76 in the sequence thr-asn-cys-tyr-glu-ser-tyr-serthr-metser; tyr-92 and tyr-97 in the seser-ser-lys-tyr-pro-asp-cys-ala-tyrquence lys-thr-t#hr; tyr-115 is in the sequence pro-t,yr-val-pro. Temperature Perturbatum Diffewnce Spectra The decreasing magnitude of Aetp for ATA with increasing SDS concentration supports the idea tha,t it is the chromophore-Hz0 interaction which generates Ae6tp, because Aq, decreases with increasing interaction between the chromophore and SDS. The ratios of the AetP values for RNase and RCM-RNase in SDS are close to unity. As, for RNase is constant in O-53 mM SDS. This may be explained as: (1) no change in the conformation of RNase; (2) disruption of the native conformation of RNase with equivalent burial in a new conformation; or (3) counterbalancing effects of externalization and masking. The first of these is ruled out by other data (Ae, , CD, and fluorescence). We cannot decide between the
295
second and third explanations from AQ, data alone. Explanation 2 disagrees with ,,except for the possibility of burial in a A~D new conformation with AeD # 0. Both 2 and 3 require gross disorganization of RNase by SDS and are incompatible with the idea of little or no change. Explanation (3) is consistent with Acsp because the interaction of the models with SDS decreases A%, by 50 %; if this represent,s 50 % masking from solvent, we expect a similar decrease in Ae,, . We find a 45 % decrease for RCM-RNase and 34 % for ATA. At higher SDS concentration the decrease for ATA would probably be greater. Thus, the constancy of Aetp for RNase is consistent with a doubling of external tyrosyls (from 3 to 6) with concomitant halving of Ae,, per tyrosyl through masking. For RNase in water Xt, is about 3 nm smaller than those of RCM-RNase and ATA. The latter two have similar ht, values and the ratio of their Actp values is 6.2, supporting the idea that the tyrosyls of RCMRNase are exposed in HzO. The difference in Xt, between RNase and the models in H20 may arise from difference in hydration or water structure caused by groups close to tyrosyl. This might be expected to affect Aetp . But the number of external tyrosyls for RNase in Hz0 calculated from AC,, is in agreement with X-ray results and solvent perturbation results. If water structure and hydration of tyrosyls are affected by nearby groups, solvent perturbation should also be affected. Comparison of AeD , Aesp, and ACQ We have indicated that all three types of difference spectra are compatible with the hypothesis of 6 external, masked tyrosyls. But there are other compatible hypotheses. In Table V we show the AC values calculated for all possible distributions of tyrosyls, considering only integral numbers, and omitting those with 4 or more buried tyrosyls which give Ae values far from experimental (e.g., all AeD are positive). The experimental values are also given in Table V. The calculated Ae values were obtained on the following bases. For A+:AeD of exposure = -900 (one-third of - 2700) and At, of masking one exposed tyrosyl is f300
296
PITTZ
AND
(the average of RCM-RNase, lys-tyr-lys, and tyramine in 150 mM SDS adjusted upward by 2 % to give a round number). For Acsp/eM, we used the average value for exposed tyrosyl in Hz0 from RCM-RNase and all three tripeptides, and for masked tyrosyl we used the average for RCM-RNase, lys-tyr-lys, and tyramine in 150 mM SDS. In both cases20 % sucrose was the perturbant. There is a difficulty in using AE,*/E~ , in that, Q for native protein includes contributions from phenylalanine (small), disulfides and the enhanced E of buried tyrosyls. Thus comparison of AesP/eM for RNase with those of models contains an error, usually not, important, from these sources. We have compromised on the use of r, = 9000 for RNase, about midway between Edfor RNase and RCM-RNase. (In Table II we have used the actual Ed, or more accurately the observed Asp/AM.) Therefore the experimental AcsP/eXin Table V for RNase differs slightly from that in Table II. For Aebpin Hz0 we used the average of RCM-RNase and ATA, and in 53 mM SDS, the value for RCM-RNase. Table V shows that for three cases only do all three difference spectral methods give agreement,, examples 12, 17, and 18; and 18 (6 external, masked tyrosyls) gives the best agreement with experiment. Even if AetPis set aside as a relatively unproved technique, we find agreement between AE, and AesPonly for 12, 17, and 18. The agreement between Aesp , AeD, and As, supports the validity of Ace . In Table V, we have not tried to refine the results by the use of fractional residues. Instead we have allowed a range of AEvalues sufficient to account for some fractional residue in each category. The use of Ae, = - 2700 for total exposure may really repre-
sent only 2.5 tyrosyls. This would change the results somewhat, without changing the conclusions. There are other difficulties, e.g., the meaning of AC, of f300 for masking in SDS compared with +500 in HTAC, and the question of the adequacy of the models. For these reasons the main use of Table V is to indicate those distributions which are grossly incompatible with experiment, and those which are in the range of interest. Table V demonstrates the importance of using several methods.
BELL0 TABLE
V
COMPARISON OF EXPERIMENTAL A.& FOR RNASB IN SDS WITHTHEORETICALUI'SFORDIFFERENT COMBINATIONS OF BURIED, MASKED, AND ExPOSED TYROSYLB
T ES-PI e no.
Number of tzdsyls
external
EXpoSd -
Calcuhtcd spectral data
L\cD
--
1 2 3 4 5 6 7 8 9 10 11 12* 13 14 15 16 17* 1f3* 19 20 21
Experimental 3 3 3 3 4 4 4 5 5 2 2 2 1 1 1 1 0 0 0 0
0 3 2 1 0 1 2 0 1 2 3 4 2 3 4 5 6 5 4 3
AE’S
3 0 1 2 2 1 0 1 0 2 1 0 3 2 1 0 0 1 2 3
$600
0.017 0.017 0.026 0.023 0.020 0.023 0.026 0.029 0.029 0.032 0.017 0.020 0.023 0.011
0 -600 - 1200 -900 -300 +300 $900
0.017 0.020 0.017; 0.014; 0.0111 0.008~
-1000 0 - 1800 -1800 -600 -900 -1500 -2100 - 1800 -2400 -300 -900 - 1500
0.014
-185 -166 -245 -225 -196 -8’90 -250 -280 -275 -305 -170 -200 -230 -115 -145 -176 -206 -180 -160 -120 -90
- a Italicized numbers are in agreement with experimental within about 20%. Examples with * are those for which AND , AcSp , and Aetp agree.
Further Comments on Masking
Above pH 12 RNase and RCM-RNase show irreversible spectral changes arising from reaction of hydroxide ion at the disulfide and carboxymethylcysteinyl groups. Conversion of cystine to cysteic acid inhibits this effect (45). In 30 mM hexadecyltrimethylammonium chloride RNase shows the irreversible spectral effect below pH 10 (26). The low pH for this reaction in the cationic detergent suggests that cationic groups are close to the hydrophobic cystine residues. Dyson and Noltmann (20) studied the exposure of chromophores of phosphoglucose isomerasein SDS, without SDS in the model systems. They also found that the tyrosyls of this enzyme are titrated at higher pH in SDS than in water (46). They consider masking by SDS, but, conclude that if the
RNaae
AND
TYROSYL
spectra are affected, the “conclusions would still be qualitatively correct, although the numerical values for the exposed residues might differ somewhat” (20). Our data suggest that the numerical values of external tyrosyls may differ greatly. In conclusion, we have presented results that can be explained consistently on the hypothesis of disruption of the conformation of RNase with externalization and masking of tyrosyl residues. It is clear that the usual models, ATA and N-acetyltyrosine esters, are not useful at low to moderate detergent concentrations. Masking, or association of the apolar tail of a detergent with chromophores has been justifiably applied to red shift’s at low concentrations of detergents [e.g., Refs. (21), (47), (48)]. Our point is that if the red shifts of appropriate model compounds are not considered, we cannot attempt to interpret blue shifts. A blue shift means exposure, but it is probable that this is usually accompanied by some degree of masking of both normally and newly external tyrosyls. At low detergent concentrations, at which a few detergent molecules are bound, it is probable that in many cases there is no externalization and the red shift represents masking only. But in some cases there may be externalization with a net red shift arising from a greater amount of masking than externalization. REFERENCES 1. BIGELOW, C. C., AND SONENBERG, M., Biochemistry 1, 196 (1962). 2. HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Sol. Chem. 243, 2123 (1968). 3. SELA, M., WHITE, F. H., JR., AND ANFINSEN, C. B., Science 136, 691 (1957). 4. SELA, M., WHITE, F. H., JR., AND ANFINSEN, C. B., Biochim. Biophys. Acta 31,417 (1959). 5. BELLO, J., Biochemistry 8, 4542 (1969). 6. BELLO, J., Biochemistry 8, 4550 (1969). 7. CANE, M., Fed. Proc. Fed. Amer. Sot. Exp. Biol. Abstr. 28, 2 (1969). 8. LASKOWSKI, M., JR., LEACH, S. J., AND SCHERAGA, H. A., J. Amer. Chem. Sot. 82, 571 (1960). 9. LEACH, S. J., AND SCHERAQA, H. A., J. Amer. Chem. Sot. 82, 4790 (1960). 10. HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Biol. Chem. 237, 2481 (1962). 11. COWGILL, R. W., Biochim. Biophys. Acta 120, 196 (1966).
MODELS
IN
SDS
297
12. COWGILL, R. W., Arch. Biochem. Biophys. 104, 84 (1964). 13. KUNITZ, M., J. Biol. Chem. 164, 563 (1946). 14. BIGELOW, C. C., J. Biol. Chem. 236, 1706 (1961). 15. SARFARE, P. S., AND BIGELOW, C. C., Can. J. Biochem. 46, 651 (1967). 16. SELA, M., AND ANFINSEN, C. B., Biochim. Biophys. Acta 24, 229 (1957). 17. SIMMONS, N. S., AND GLAZER, A. N., J. Amer. Chem. Sot. 89, 5040 (1967). 18. JIRGENSONS, B., AND CAPETILLO, S., Biochim. Biophys. Acta 214, 1 (1970). 19. RAY, A., REYNOLDS, J. A., POLET, H., AND STEINHARDT, J., Biochemistry 8, 2606 (1966). 20. DYSON, J. E. D., AND NOLTMANN, E. A., Biochemistry 8, 3544 (1969). J. A., HERBERT, S., AND 21. REYNOLDS, STEINHARDT, J., Biochemistry 7, 1357 (1968). 22. COWGILL, R. W., Biochim. Biophys. Acta 140, 37 (1967). 23. COWGILL, R. W., Biochim. Biophys. Acta 133, 7 (1967). 24. COWGILL, R. W., Arch. Biochem. Biophys. 100, 36 (1961). 25. BIGELOW, C. C., J. Theor. Biol. 16,187 (1967). 26. BELLO, J., Arch. Biochem. Biophys., submitted for publication. 27. SIMPSON, R. T., AND VALLEE, B. L., Biochemistry 6, 2531 (1966). SCATTURIN, A., AND 28. TAMBURRO, A. M., MORODER, L., Biochim. Biophys. Acta 164, 583 (1968). 29. PFLUMM, M. E., AND BEYCHOK, S., J. Biol. Chem. 244, 3973 (1969). 30. HORWITZ, J., STRICKLAND, E. H., AND BILLUPS, C., J. Amer. Chem. Sot. 92, 2119 (1970). M. E., AND 31. WOODY, R. W., FRIEDMBN, SCHERAGA, H. A., Biochemistry 6, 2034 (1966). 32. YANARI, S., AND BOVEY, F. A., J. Biol. Chem. 236, 10 (1960). 33. REYNOLDS, J. A., AND TANFORD, C., Proc. Nat. Acad. Sci. U.S.A. 66, 1002 (1970). 34. REYNOLDS, J. A., AND TANFORD, C., J. Biol. Chem. 246, 5161 (1970). 35. FISH, W. W., REYNOLDS, J. A., AND TANFORD: C., J. Biol. Chem. 246, 5166 (1970). 36. PITT-RIVERS, R., AND IMPIOMBATO, F. S. A., Biochem. J. 109, 825 (1968). 37. HAMAGUCHI, K., AND KURONO, A., J. Biochemistry Tokyo 64, 111 (1963). 38. GRATZER, W. B., AND BEAVEN, G. H., J. Phys. Chem. 73, 2270 (1969). 39. HERSKOVITS, T. T., AND L~SKOWSKI, M., JR., J. Biol. Chem. 236, 11 PC 56 (1960). 40. HARRINGTON, W. F., AND SELA, M., Biochim. Biophys. Acta 31, 427 (1959).
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41. HARRINGTON, W. F., AND SCHELLMAN, J. A., C. R. Trav. Lab. Carlsberg 50, 21 (1956). 42. KLEE, W. A., Biochemistry 6, 3736 (1967). 7, 2731 (1968). 43. KLEE, W. A., Biochemistry 44. TANFORD, C., J. Amer. Chem. Sot. 84, 4240 (1962). 45. TRAMER, Z., AND SHUGAR, D., Acta Biochim. Polon. 6, 235 (1959).
BELL0 46. DYSON, J.E.D., AND NOLTMANN, E. A., Biochemistry 8, 3533 (1969). 47. ZAKREZEWSKI,K., AND GOCH,H., Biochemistry 7, 1835 (1968). 48. STEINHARDT, J., AND REYNOLDS, J. A., “Multiple Equilibria
in Proteins,”
demic Press, New York,
1969.
Chap. 8. Aca-
OF
RIOCHEMISTRY
Interaction
AND
RIOPHYSICS
of Sodium
147, 284298
(1971)
Sulfate
with
Dodecyl
in Ribonuclease
A and EUGENE
Department
of Biophysics,
State University
Model
Tyrosyl
Chromophores
Compounds’
P. PITTZ2 of New York at Buffalo,
Bu$alo,
New York
14.!314
AND
JAKE Department
of Biophysics, Received
Roswell
BELL0
Park Memorial
May 5, 1971; accepted
Institute, July
Buffalo,
New York 1,$.203
19, 1971
Ultraviolet difference spectra, solvent perturbation difference spectra, and temperature perturbation difference spectra indicate that tyrosyl residues of model compounds are affected by sodium dodecyl sulfate. This effect is dependent on the nature of the model compound, being enhanced by positive charges, and is attributed to partial masking of the tyrosyl chromophores by sodium dodecyl sulfate. With reduced carboxymethylated ribonuclease as a model, all three difference spectral methods can be interpreted as indicating nearly complete externalization of tyrosyl chromophores in ribonuclease in the detergent. With small tyrosyl model compounds the calculated number of external tyrosyl residues depends on the nature of the model compound. Using net positively charged tyrosyl compounds as models, nearly 6 external tyrosyl residues are calculated for RNase. N-Acetyltyrosine amide or N-acetyltyrosine esters appear to be inadequate models for tyrosine in proteindetergent solutions because of their weak int,eractions with detergents.
Reports of the state of the tyrosyl residues in RNase (polyribonucleotide 2-oligonucleotide-transferase, cyclizing) in the presence of sodium dodecyl sulfate (SDS3) are dis1 This work was supported by grants from the Institute of General Medical Sciences of the National Institutes of Health (GM 13435 to Roswell Park Memorial Institute, and Training Grant GM 00718 to the Department of Biophysics, State University of New York at Buffalo) and from the National Science Foundation (GB 7523 and GB 20083). Th is work is abstracted from a dissertation by E.P.P., State University of New York at Buffalo, 1970. 2 Present address: Graduate Department of Biochemistry, Brandeis University, Waltham, MA 02154. 3 Abbreviations used: RNase, bovine pancreatic ribonuclease, EC 2.7.7.16; SDS, sodium dodecyl sulfate; CD, circular dichroic spectrum; ATA. N-acetyltyrosine amide; RCM-RNase, RNase having its disulfides reduced and carboxymethyl-
cordant. Bigelow and Sonenberg (1) have shown that SDS (> 10 mM) causes a blue shift of the tyrosyl band of RNase with a Ae of -750 indicating exposure of about one additional tyrosyl residue. Herskovits and Laskowski (a), studying the solvent perturbation difference spectrum of RNase in 150 mM SDS, found either no change or a slight decrease in the fraction of accessible tyrosyls depending on the perturbant used. Since there is uncertainty concerning the state of the tyrosyls in RNase in the presence of SDS, we have studied the effect of SDS on the spectral properties of tyrosyl chromophores of RNase and of several model compounds. It appeared important in this type of study to investigate more adequately the effect of SDS on a variety of models varying ated; TP, thermal turbation. 284
perturbation;
SP, solvent
per-
RNase
AND
TYROSYL
in charge and hydrophobic character. Other investigators have omitted SDS from their model systems on the ground that SDS does not affect the chromophores at low SDS concentration. In this communication we present evidence that SDS does affect tyrosyl and tryptophyl chromophores even at rather low SDS concentrations, and that the effect is very strongly dependent on the model compound selected. MATERIALS
AND
METHODS
Materials Ribonuclease A was purchased from Mann Research Laboratories, New York, NY, as were N-acetyl-L-tyrosine amide, gly-tyr-gly, leu-tyrleu, lys-tyr-lys, and glu-tyr-glu. Tyramine hydrochloride was purchased from Calbiochem and Eastman Orga,nic Chemicals. RCM-RNase was prepared by the method of Sela et al. (3,4), except that Cleland’s reagent (dithiothreitol) was used instead of thioglycolic acid. Circular dichroism measurements gave no evidence of a band at 280 nm in RCM-RNaae, indicating loss of native conformation around the tyrosyls. The molar extinction coefficient of this derivative was 8350 at 274.2 rnp using a molecular weight of 14,272. The molar extinction coefficients of RNase and ATA at the 274-278 nm peak were 9650 and 1400, respectively. The coefficients of leu-tyr-leu, lystyr-lys, glu-t,yr-glu, and tyramine were, respectively, 1400, 1320, 1420, and 1450. A value of 1400 was assumed for gly-tyr-gly, but this material contained 1&20y0 of an iodinatable impurity (Dr. 0. A. Roholt, personal communication). The concentrations of these derivatives were estimated from the molar extinction coefficients. Glutathione (oxidized) was purchased from Nutritional Biochemicals Corp., Cleveland, OH. Sodium dodecyl sulfate and hexadecyltrimethylammonium chloride (HTAC) were purchased from Eastman Organic Chemicals and were used without purification. Several experiments were repeated with Sequanal Grade SDS from Pierce Chemical Co., with no difference in results. Urea, sucrose, and sodium acetate were reagent grade. Urea solutions were deionized with MB-3 resin immediately before use. Spectral
Investigations
The temperature perturbation difference spectral method has been developed independently by Bello (5, 6) and Cane (7). Thermal perturbation (TP) difference spectral measurements were carried out as described earlier (5), except that the temperatures used were 27 and 6”.
MODELS
IN
SDS
285
Ultraviolet difference spectra were measured in a Cary 15 spectrophotometer at 27”. The four-cell method of Laskowski et al. (8) was employed to compensate for solvent. For gly-tyr-gly and leutyr-leu the two-cell method was employed, and the difference spectra were corrected for light scattering by the method of Leach and Scheraga (9). Both methods were used for lys-tyr-lys and glu-tyr-glu, the results agreeing within 370. Solvent perturbation (SP) difference spectra were obtained by the method of Herskovits and Laskowski (10). The sample and reference were thermostatted at 27’. There was some difficulty in obtaining flat base lines in a few cases because of very slight differences in SDS concentrations and was easily compensated by correcting for light scattering (9), or by slight adjustments in the SDS concentration in the chromophore-free reference cells. Circular dichroism measurements were taken at 25 and at 4” with a Jasco ORD/5 recording spectropolarimeter with CD accessory. When spectra were taken at 4”, nitrogen gas was used to purge the sample compartment of water vapor. Fluorescence measurements were made at 25” with an Aminco-Bowman spectrophotofluorimeter at an excitation wavelength of 275 nm; the emission was scanned to 500 nm. The slit arrangement, was No. 3. Quantum efficiencies were calculated on the basis of & = 0.014 for ribonuclease A in water at pH 5.8 (11, 12). The fluorescence of SDS was negligible. Enzymatic activity measurements were made by the method of Kunita (13). RESULTS
Ultraviolet Di$erence Spectral Measurements Ultraviolet difference spectral measurements for RNase and RCM-RNase in 30 mM SDS 2)s. RNase and RCM-RNase, respectively, in water are shown in Fig. 1. For solubility, RCM-RNase was studied at pH 2.6 while RNase was studied at’ pH 5.8. No difference spectrum occurs for RNase in 30 mM SDS at pH 5.8 vs. RNase in 30 mM SDS at pH 2.6, indicating that SDS effects on the tyrosyl chromophores in RNase are the same at these pH values. RNase in 30 m&x SDS vs. RNase in water gives a negative difference spectrum similar to that of Bigelow and Sonenberg (1). However, Fig. 1 shows a difference spectrum for RCM-RNase in 30 m&f SDS vs. RCM-RNase in water with a positive AE~ of 1620 at 287 nm, where
286
PITTZ
AND
BELL0
D indicates the classical ultraviolet difference spectrum, as distinguished from the SP and TP difference spectra discussed below. Figure 2 shows positive difference spectra for lys-tyr-lys and tyramine in 150 rnmrSDS vs. these models in aqueous solution. RCMRNase in 150 rnM SDS gives a positive difference spectrum similar to that for RCMRNase in 30 mM SDS. The difference spectral results are summarized in Table I. The AeD values for the tyrosyl model compounds, except for RCMRNase, are 6 times the AcI, measured, for comparison with RNase and RCM-RNase, which contain 6 tyrosyl residues. AeD is negaF’ tive for RNase at all SDS concentrations. RCM-RNase, ATA, gly-tyr-gly, leu-tyr-leu, A jrnp lys-tyr-lys, and tyramine give positive Fro. 2. The uv difference spectra of model comAE~ values. AQ for glu-tyr-glu, which has a pounds in 150 mM SDS 8s. these solutes in water. net negative charge at pH 5.8, is effectively 1, lys-tyr-lys (6.35 X lo-+ M), pH 5.8; 2, tyramine zero in 30 and 150 rn>r SDS. (1.1 X 10e3 M), pH 5.8; 3, RCM-RNase (2.82 X We shall assume here that SDS enfolds lo-* M or 1.7 X lo-” M in tyrosyl residues), pH 2.6. the tyrosyl chromophore, part,ially masking Each unit on the AOD scale represents 0.05 OD. it from other components of the solvent. The O-l slidewire was used. There is the alternate possibility that SDS does not enfold the tyrosyls, but causes them This is not possible for tyramine but is posto be buried within the protein or peptide. sible for protein and partially so for the tripeptides. We shall consider the folding hypothesis in the Discussion; here we shall lsee the conclusions reached on the hypothesis of masking by SDS. We shall use the definif tions: buried, enfolded within the protein; masked, shielded by SDS from other solvent components; exposed, outside the protein and not masked; external, outside the protein, whether masked or not. By subtracting AQ, for RCM-RNase, or 6 AQ, for small models, from Ae, for RNase we compensate for the masking of the tyrosyl by SDS. This is the same type of correction that has been made for other denaturants such as urea and LiBr (14, 15). Column 4 of Table I shows these corrected AeD values. Fully denatured RNase has a AE~ of about -2700 relative to native RNase (14). If this -I represents exposure of all of the buried FIG. 1. The uv difference spectra of RNase and tyrosyls upon denaturation, we can calcuRCM-RNase in 30 mu SDS vs. these solutes in late the number of external tyrosyls in water; (a), RNase (7.7 X 10e5 M) in 30 mM SDS vs. RNase in SDS solution (Table I). The calRNase in water, pH 5.8; (b), RCM-RNase (6.1 X culation depends on the chosen model. For W5 M) in 30 mM SDS vs. RCM-RNase in water, lys-tyr-lys in 30 rnbr SDS Ae, is about the pH 2.6. Each unit on the AOD scale represents 0.01 same as in 150 ml\r SDS. For ATA Ae, is unit OD.
RNase AND TYROSYL
MODELS
TABLE THE uv DIFFERENCE
IN SDS
I
SPECTRAL RESULTS FOR RNASE, RCM-RNASE, vs. THESE SOLUTES IN WATER
Solute”
RNase, 10 mM SDS RCM-RNase, 10 mM SDS ATA, 10 mM SDS RNase, 20 mM SDS RCM-RNase, 20 mM SDS ATA, 20 mM SDS RNase, 30 mM SDS RCM-RNase, 30 mM SDS ATA, 30 mrvr SDS Gly-tyr-gly, 30 mM SDS Leu-tyr-leu, 30 mM SDS Glu-tyr-glu, 30 mu SDS Lys-tyr-lys, 30 mM SDS RNase, 52.5 rnx SDS RCM-RNase, 52.5 mM SDS ATA, 52.5 mM SDS RNase, 150 mru SDS RCM-RNase, 150 mM SDS Lys-tyr-lys, 150 mM SDS Tyramine, 150 mM SDS Glu-tyr-glu, 150 mM SDS
Aebsc D
-920 +1450
+6’3
(+lO)
-860 +1570
+120
287
(+20)
- 1010 +1620 +294 (+49)
+216 (+36) +492 (+82) -36 (-6) +1632 (f272) -980 +1570
+480 (+80) - 1010 +1840 +1674 (f275) +1790 (+298)
+36 (+6)
AND MODEL PEPTIDES
AD b-d
AED (corrected)a
286.9 285.5 284.7 286.3 285.6 284.7 286.2 285.8 284.8 284.0 285.1 284.0 284.6 286.8 285.8 284.6 286.5 285.5 284.7 284.5 284.0
-2370 -980 - 2430 -980 - 2630 - 1304 - 1226 - 1502 -974 - 2724 - 2550 - 1460 - 2850 -2684 -2800 - 1046
IN
SDS
5.7 4.3 5.7 4.2 5.9 4.6 4.5 4.8 4.3 6.0 5.9 4.9 6.1 6.0 6.1 4.4
= RCM-RNase is at pH 2.6. All other solutes are in 0.01 M sodium acetate buffer, pH 5.8. b The AND listed here for ATA, gly-tyr-gly, leu-tyr-leu, lys-tyr-lys, and glu-tyr-glu and tyramine are 6 times the Aeo actually measured. This enables us to compare ANDfor RNase with AEDfor the model compound, since RNase contains six tyrosyl molecules. The number in parentheses is the observed A6n . c The difference spectra of gly-tyr-gly and leu-tyr-leu are corrected for light scattering by the method described by Leach and Scheraga (9). This was necessary since only two cells were used to obtain difference spectra,. d The difference spectral data are not corrected for the effect of SDS on disulfide absorbance since a AED of only 2 was found for oxidized glutathione in 30 mu SDS vs. glutathione in water. Even if glutathione is not a perfect model the error must be negligible. c The number of exposed tyrosyl residues listed is based upon the fact that a AEDof -2700 represents the exuosure of all of the buried tvrosvl residues of RNsse. A~D (corrected) is ANDfor RNase minus Ac, for the model for which the value is shown.
small in 10 mM SDS and increases with increasing SDS concentration. Ae, values for RNase and RCM-RNase in 10 rnbr SDS reach 90 and SO%, respectively, of AED in 150 mM SDS, indicating that the effect of SDS on the tyrosyl chromophores of these proteins is nearly complete at 10 mM SDS. (Possibly Ace would be still larger and masking more complete at much higher SDS concentrations.) We have also measured difference spectra for RNase, glu-tyr-glu and tyramine, at pH 5.8, in 30 m&r hexadecyltrimethylam-
monium chloride vs. these solutes in water. Glu-tyr-glu gives AcD of +500 (3000 for 6 AED) at 286 nm while RNase and tyramine had AcD of +30 and +60, respectively. The CD spectra of RNase in water and in this detergent were the same at 240-300 nm. Solvent Perturbation Dierence Spectra The SP difference spectra for RNase and RCM-RNase in water and 150 mM SDS with sucrose as perturbant are shown in Fig. 3. SDS (150 mM> depresses the SP spectrum of RNase, and even more so, that
288
PITTZ
AND BELL0
of RCM-RNase. Figure 3c shows the depression of the SP spectrum of lys-tyr-lys in 150 mM SDS as compared to this model compound in water.
kmr FIG. 3. Solvent perturbation
A, w
difference spectra in water and 150 lll~ SDS. a: 1, RNase (2.4 X lo-’ M) in water, pH 5.8; 2, RNase (2.4 X lo-* M), in 150 m SDS, pH 5.8; b: 1, RCM-RNase (2.8 X low4 M), in water, pH 2.6; 2, RCM-RNase (2.8 X 1V4 M), in 150 lll~ SDS, pH 2.6; c: lys-tyr-lys (1.2 X 10-’ M); 1, water, pH 5.8; 2, in 150 mM SDS. For RNase and RCM-RNase 20% sucrose is used as perturbant. For lys-tyr-lys, 10% sucrose is used as perturbant. Each unit on the AOD scale represents 0.01 unit OD.
Figure 4a indicates that Acsp/ena vs. sucrose concentration is linear for RNase and model compounds in aqueous solution up to 20% sucrose, and that the slope for RNase is about 0.55 t’hat of the others, indicating the fractional exposure of tyrosyls. In 150 mM SDS (Fig. 4) only glu-tyr-glu shows linear behavior, with slope identical to that in wat’er, while RNase and the other models show nonlinearity, the deviation being greater for lys-tyr-lys and tyramine than for RNase and RCM-RNase. The deviation is reproducible. Thus the calculated exposure of tyrosyls depends not only on the model compound but also on the concentration of perturbant, especially in multicomponent systems. The significance of the nonlinearity will be considered in the Discussion. Table II summarizes the results of the SP difference spectral method. Column 5 shows the fraction of external tyrosyl residues calculated by dividing A+,/E~ for RNase by AGJQ for the model compound at the same SDS and perturbant concentration. Column 6 gives the fraction of external tyrosyl residues of RNase multiplied by 6, to give a quantity which we propose is equal to the number of external tyrosyl residues of RNase. 2.9-3.5 tyrosyl residues are found to be exposed (= external in this case) for RNase in aqueous solution. For RNase in 150 m&T SDS the range of values is 4.4-6 external tyrosyl residues, except with glu-
FIG. 4. Aesp/c~ vs. sucrose concentration. (a), in water; (b), in 150 rnM SDS. 0, glu-tyr-glu; n, tyramine; 0, lys-tyr-lys; 0, RNase; & RCM-RNase. The horizontal scale is offset to accommodate all the plots.
RNase
AND
TYROSYL TABLE
SUMMARY
MODELS
289
SDS
II
OF SOLVENT PERTURBATION DIFFERENCE SPECTRAL RESULTS FOR RNASE COMPOUNDS IN WATER AND 150 mM SDS USING 10 AND 20% BY WEIGHT SUCROSE AS PERTURBANT % Sucrose perturbant
system
RNase,
IN
Fraction of external tyrosinesC
AND MODEL
Fraction X6O
Hz0
0.010
236.0
10
-
-
RCM-RNaae, H20 Lys-tyr-lys, Hz0 Tyramine, Hz0 Glu-tyr-glu, Hz0 RNase, 150 mM SDS
0.018 0.020 0.021 0.021 0.006
284.5 284.5 284.0 284.5 285.5
10 10 10 10 10
0.55 0.50 0.48 0.48 -
3.3 3.0 2.9 2.9 -
RCM-RNase, 150 mM SDS Lys-tyr-lys, 150 mm SDS Tyramine, 150 mu SDS Glu-tyr-glu, 150 mu SDS RNase, Hz0
0.007 0.006 0.007 0.021 0.021
284.5 236.0 236.0 234.5 286.0
10 10 10 10 20
0.9 1 0.9 0.3 -
5 6 5 2
RCM-RNase, Hz0 Lys-tyr-lys, Hz0 Tyramine, Hz0 Glu-tyr-glu, Hz0 RNase, 150 mM SDS
0.036 0.038 0.041 0.039 0.016
284.5 284.5 284.0 284.0 285.0
20 20 20 20 20
0.58 0.55 0.51 0.54 -
3.5 3.3 3.1 3.2 -
RCM-RNase, 150 mra SDS Lys-tyr-lys, 150 mM SDS Tyramine, 150 mM SDS Glu-tyr-glu, 150 mM SDS
0.016 0.020” 0.022 0.039”
284.5 286.0 285.0 284.5
20 20 20 20
1.0 0.80 0.73 0.41
6.0 4.8 4.4 2.5
a eM is always measured in aqueous solution without SDS or perturbant. h x,, is rounded to the nearest 0.5 rnr. c Each value shown is for RNase, using as model the compound for which
tyr-glu as model. For glu-tyr-glu the calculated number of external tyrosyls is similar to the number of exposed tyrosyls calculated by Herskovits and Laskowski (2). This is to be expected since these workers did not use SDS in their model system and glu-tyr-glu appears not to interact with SDS. With 20% sucrose as perturbant A~,,/Q for RNase in water is 0.021, in agreement with the value of 0.020 of Herskovits and Laskowski (2). For RCM-RNase we found 0.036 compared with 0.043 of these workers. In 150 mM SDS we found 0.016 for RNase compared with 0.020 of Herskovits and Laskowski (2). Temperature Perturbation Difference Spectral Results The TP difference spectra of RNase and RCM-RNase in the presence and absence of 30 mM SDS are shown in Fig. 5. From the negative extremum (at 286-290 nm) in each of the curves Ae,, is calculated, and the
-
the value is given.
ratio of Actp for RNase to Aetp for the model is used to estimate the external t,yrosyl residues. Figure 6 shows that Aetp for RNase in 30 mM SDS and RCM-RNase in water and in 30 mM SDS is linear with temperature, indicating the probability of no significant conformational transition affecting Actp over this temperature interval. Earlier (5) it was shown that Ae,, of RNase in water is linear from 4 to 25”. Additional support for the absence of a major conformational change is that exposure or burial of a single tyrosyl produces a AE about 3-6 times as great (depending on wavelength) as Ae,, for the entire RNase molecule. This would dominate the spectrum, giving essentially a denaturation or renaturation spectrum (6). Table III summarizes the TP spectral data. The Act* values are not corrected for the small (5) contribution from buried or masked chromophores arising from the volume change on cooling. The decreasing Act, for ATA with increasing SDS concen-
PITT2
AND BELL0
compound, and 3.5 when ATA is the model. For RNase in 8 M urea, 5.6 tyrosyl residues are found exposed; this probably represents real burial because in urea the number of exposed tyrosyls is pH dependent, becoming 5.9-6.1 at pH 4-5. Ae,, of RNase is constant in O-53 mM SDS; Aetp of RCM-RNase changes on transfer in O-10 mM SDS and remains constant in lo-53 mM SDS; Act,, for ATA does not change on transfer in O-10 mM SDS but changes continuously (with decreasing slope) in lo-53 mM SDS. FIQ. 5. The TP difference spectra. Spectrum 1, RNase (7.3 X 10-6 M) in 0.01 M sodium acetate buffer, pH 5.8; 2, RNase (7.7 X 1OU5M) in 30 mu SDS, 0.01~ sodium acetate buffer, pH 5.8; 3, RCMRNase (6.1 X low6 M), pH 2.6; 4, RCM-RNase (6.1 X lO-&M) in 30 nm SDS, pH 2.6. Each unit on the AOD axis represents 0.01 unit of OD. Zero AOD for each spectrum is the horizontal long wavelength region.
tJ 0
0
6
IO
Fluorescence Studies Relative fluorescence emission values, Q/&o, were calculated for RNase and RCM-RNase in SDS relative to water. The data in Table IV indicate that the quantum efficiencies of both RNase and RCM-RNase increase on transfer from water to SDS solutions. The RFE values for RNase and RCM-RNase are almost constant in 2-53 mM SDS indicating that the entire effect is observed at the lower detergent concentration. Enzymatic Activity RNase in 8 M urea was 80% as active as in water. It is known that RNase in urea refolds in the presence of substrates (16). When 30 m&l SDS was added to RNase in water or in 8 M urea there was no spectral evidence of enzymatic activity even after 24 hr of incubation with substrate.
14
TEMP.
16
22
26
*C
FIG. 6. Act, as. temperature. 0, RNase in 30 rnM SDS; n , RCM-RNase in water; and 0, RCMRNase in 30 mM SDS. The base line was set at 27”.
tration indicates that Ae,, for masked tyrosyls is smaller than for exposed tyrosyls. Column 5 of Table III lists ratios of Actl, for RNase to A+, for RCM-RNase and for ATA in the same solvent system. Provisionally we assume that only chromophores exposed to water give Aetp , and that this ratio represents the fraction of external tyrosyl residues in RNase. This will be considered in the Discussion. For RNase in water, 3.4 t)yrosyl residues are exposed to solvent when RCM-RNase is the model
Circular Dichroism (CD) and Transition Temperature Measurements For RNase in water and 20% sucrose identical spectra were found between 210 and 310 nm. In the 240-310-nm region the spectra were similar to those found by Simmons and Glazer (17) both in water and in 20% sucrose. EL - ER at 280 nm was reduced by 87 % for RNase in 150 mM SDS (in agreement with Simmons and Glazer). For both RNase and RCM-RNase in SDS the CD spectrum in the 210-220-nm region indicated an increase in helical and/or /3structure in agreement with the findings of Jirgensons and Capetillo (18). In regard to the TP spectra, the CD spectrum of RCM-RNase in water and 20 % sucrose was
RNase AND TYROSYL TABLE
MODELS
IN SDS
291
III
SUMMARY OF TP DIFFERENCE SPECTRAL RESULTS ON RNASE, RCM-RNASE ATA IN WATER, 8 M UREA AND SDS
AND
Solute RNase, water RCM-RNase, water ATA, water RNaae, 8 M urea RCM-RNase, 8 M urea ATA, 8 M urea RNase, 10 mu SDS RCM-RNaae, 10 mM SDS ATA, 10 mM SDS RNase, 20 mu SDS RCM-RNaae, 20 mM SDS ATA, 20 mM SDS RNase, 30 mu SDS RCM-RNase, 30 mM SDS ATA, 30 mM SDS RNase, 52.5 mM SDS RCM-RNaae, 52.5 mu SDS ATA, 52.5 mnd SDS
-198 -328 -318 -308 -320 -312 - 198 -181 -318 - 194 -184 -246 - 199 - 180 -228 -196 -181 -210
(-53) (-52) (-53)
(-41) (-38) (-35)
-184 -328 -318 -296 -320 -312 - 185 -181 -318 - 185 -181 -246 - 185 -181 -228 -185 -181 -210
289.5 286.3 286.2 288.0 287.4 287.0 289.0 288.9 286.2 289.2 289.5 286.5 289.1 2239.4 286.9 289.1 289.3 287.0
0.56 0.58 0.93 0.94 1.02 0.58 1.02 0.75 1.02 0.81 1.02 0.88
3.4 3.5 5.6 5.6d 6.1 3.5 6.1 4.6 6.1 4.9 6.1 5.3
0 For ATA AE$,,not in parentheses is 6 times the difference in molar extinction shown in parentheses. 5 Act,, (corr) takes into account the Aetp for the four disulfides in RNase using oxidized glutathione in water (Aetp = 3.8) and in SDS (Aetp = 3.2) aa the model compound. c Number of external tyrosyls in RNase, calculated from &Q for the model indicated. d At pH 5.5, 6.0 exposed tyrosines were found, corrected for disulfides. TABLE
IV
SUMMARY OF FLUORESCENCE EMISSION RESULTS FOR RNASE AND RCM-RNASE IN WATER AND SDS
the same at 25 (with or without at 4 than at 25” was no difference syl region.
and 4“. The CD in SDS sucrose) was 10% greater at 210-215 nm, but there above 232 nm in the tyro-
Solute and solvent
DISCUSSION RNase, RNase, RNase, RNase, RNase, RNase,
water 2 mM SDS 10 rnM SDS 20 mM SDS 30 mM SDS 52.5 mM SDS
RCM-RNase, RCM-RNase, RCM-RNase, RCM-RNase, RCM-RNase, RCM-RNase,
water 8 M urea 1.0mM SDS 20 mM SDS 30 mM SDS 52.5 mM SDS
0.014 0.034 0.036 0.036 0.036 0.036
1.0 2.4 2.6 2.6 2.6 2.6
0.035 0.040 0.056 0.055 0.057 0.057
1.0 1.1 1.6 1.6 1.6 1.6
a All measurements were done at a protein concentration of 1.3 x 10e6 M.
Before considering the results we shall recall several possible states of tyrosyl residues : (1) buried inside the protein in native or nonnative conformation; (2) exposed to solvent without masking; (3) externalized and masked by SDS. The idea of masking of chromophores by detergent has been considered by several investigators [e.g., Refs. (2), (19), (ZO)], some of whom have not considered the possibility very likely. Reynolds et al. (21) studied the spectral effect of low levels of alkanoates on serum albumin and proposed the red shift to be “possibly induced by the close proximity of the hydrocarbon tail to the aromatic
292
PITTZ
AND BELL0
residues.” We agree and suggest that such an effect can be hidden in a net blue shift. Cowgill (11, 12) showed that the tyrosyl fluorescence of RNase increases in the presence of SDS, reaching a plateau at 3-10 mM SDS; Table IV shows that there is no further change up to 53 mM SDS. Our data are similar to Cowgill’s and extend to the SDS concentrations used in our other experiments. Cowgill (22) showed that reduction of the disulfide cross-links with formation of new, non-cross-linking disuljides (by oxidizing reduced RNase in the presence of a mercaptan) resulted in quantum efficiencies in 8 no urea or 5 mM SDS the same as for unreduced RNase in these solvents. He also showed that RCM-RNase gave larger quantum efficiencies than did RNase or the derivative just described. Cowgill’s results indicate that disulfides quench tyrosyl fluorescence, and that the high quantum efficiency of RCM-RNase in SDS does not necessarily mean that in SDS the tyrosyls of RCM-RNase are in much different environments than in RNase. Cowgill (22-24) showed that the peptide groups, disulfides, and carboxyl groups can quench tyrosyl fluorescence. If SDS causes burial of tyrosines we do not know whether or not to expect an increase in fluorescence because of the presence of internal quenchers. Externalization and masking of tyrosyls inside the nonpolar portion of SDS is consistent with the decreased quenching. However, titration results (26) indicate an interaction between the tyrosyl hydroxyl and the charged end of the detergent. Circular Dichroism There have been several investigations of the ORD and CD properties of RNase in SDS (17, 18, 27, 28). ORD and CD investigations in the far ultraviolet, which are related to the secondary structure, are not directly relevant to the tyrosyls. The rotatory properties at the 280 nm tyrosyl extremum are of more direct interest. Simpson and Vallee (27) attribute the 280 nm CD band to buried tyrosines. Simmons and Glazer proposed that the 280 nm CD band arises from exposed tyrosines. Pflumm and Beychok (29) and Horwitz et al. (30) consider exposed and buried tyrosyls, as well as
disulfides, to be involved. Horwitz et al. proposed that a significant portion of the CD extremum arises from the most buried tyrosyl. The nearly complete disappearance of the 280 nm extremum suggests that the conformation of RNase in SDS is so disrupted as to cause a gross change in the environment in this tyrosine. Burial in nonnative conformation is not irreconcilable with the CD data. We might expect that such buried tyrosyls would be in environments that would give rise to a CD band; this is not conclusive. State 2, exposed but not masked, is consistent with the CD data and is awkward, but not impossible, to reconcile with the AC, data, which appear to indicate that only one more tyrosyl becomes exposed; that is, the CD extremum might vanish as the result of the sum of small changes among several tyrosyls. The CD result is easily rationalized for State 3, tyrosyls externalized and masked. We propose, then, that the elimination of the 280 nm extremum of RNase by SDS can be explained by disruption of the conformation, and externalization of the tyrosyl residues. This is consistent with fluorescence and AeD . Ultraviolet Difference Spectra The fundamental question is that of the appropriateness of the model system, both as to solvent and chromophore. Bigelow and Sonenberg (1) did not compensate for the effect of SDS on model chromophores; they indicated that Yanari and Bovey (32) found only a small red shift in the spectrum of N-acetyltyrosine ethyl ester in SDS above the critical micelle concentration. Actually, AC, for acetyltyrosine ethyl ester in the 225 rniM SDS used by Yanari and Bovey was +250 at 287 nm (+ 1500 for 6 tyrosyls). While Ae, is small for ATA at low SDS concentration, such as 10 mM, it is much larger for some other models. Should the corrections shown in Table I be made? If the tyrosyls of RNase are buried and not masked by SDS, or if some are exposed and the effect of SDS is solvent perturbation similar to that of sucrose, not arising from a strong association, the correction is small or inappropriate. The data of Table I show a significant effect of SDS on some models,
RNase
AND
TYROSYL
This cannot be a simple solvent perturbation as it is as much as 100 times that of sucrose, normalized for concentration. So large an effect means a strong association, i.e., masking. For RNase, RCM-RNase, and the tripeptide models, it is conceivable that the AeD arises from folding of the molecules to bury tyrosyls. This is not possible for tyramine, which is too small for any significant degree of folding. Yet tyramine shows a A+ as large as any other. Since there is binding of SDS Do the chromophore of tyramine, it is likely that it also occurs with RNase. Therefore, we must make a correction in Ae, of RNase for the masking. The observed AQ, for RNase in SDS might arise from several combinations of exposure, masking, and burial. This will be considered further in connection with Aesp and AQ,, . In Table I using the corrections for RCM-RNase and for the positively charged models we have calculated that all of the tyrosines are external and masked. Perhaps the correction should be less than 6 AQ, for lys-tyr-lys or tyramine because some tyrosyl regions of RNase lack positive charges. But the titration data on aminoacet,ylated RNase (26) show that for the protein, in contrast to the tripeptides, positive charges are not important. The nearly zero AC, for RNase in 30 mM hexadecyltrimethylammonium chloride might have been interpreted as indicating either no effect, or complete externalization and masking of tyrosyls, i.e., about - 2700 for exposure counterbalanced by about +3000 (6 times AE~ for glu-tyrglu) for masking. But the normal CD spectrum at 260-300 nm indicates that the cationic detergent does not disrupt the conformation. In the case of SDS the disappearance of the tyrosyl CD extremum supports the interpretation of disruption of conformation with externalization and masking of tyrosyls. AQ, values for lys-tyr-lys, tyramine, and RCM-RNase are about one-third of Ae for exposure of one buried tyrosyl of RNase (14). This suggests that masking is only partial. HTAC gives a AE~ with glu-tyr-glu almost twice as large as does SDS with tyramine. If this represents more complete
MODELS
IN
SDS
293
masking, perhaps SDS can mask some tyrosyls of RNase or RCM-RNase more completely than it masks tyramine. If so, the Ae, of +1620 for 0.03 M SDS with RCMRNase might agree with 6 AQ, for tyramine because some tyrosyls of RCM-RNase have greater and some have lesser AQ, values. But the long wavelength absorption maxima of lys-tyr-lys in 30 mM SDS and of glu-tyrglu in 30 mM HTAC are the same (26). Also A+, is about only 10% greater for RCMRNase in 150 mM SDS than in 30 mM. If HTAC masks more completely it may have a greater affinity; if so, why does it seem t’o be a weaker denaturant? Reynolds and Tanford (33) have presented data which suggest that below 5 mM SDS, monomer, not micelle, binds to protein. That the effect of SDS on RNase and RCMRNase appears to be largely complete at the lower concentrations of SDS may arise from the binding being largely dependent on SDS monomer concentration, which is constant over the range studied. However, binding to ATA is dependent on total SDS concentration and, presumably, on SDS micelles. Binding of SDS to protein (at high enough concentration to cause disruption) is regarded as largely hydrophobic. Binding to small models is highly dependent on charge. This does not necessarily mean that the small models are invalid. Reynolds and Tanford (34) found that non-disulfide proteins in SDS appear to be rods of similar radius and of length proportional to molecular weight and quite unlike the proteins in the absence of SDS. If the disulfides are intact the elution position (gel chromatography) of the protein-SDS complex is different from that of the complex wit’h reduced protein (35). Reduced proteins bind about 1.4 g SDS/g protein, and unreduced proteins about 1 g/g (33, 36). Even though binding of SDS to unreduced protein is less than to reduced protein, it is highly likely t’hat the conformation of the unreduced protein is disrupted. For unreduced bovine serum albumin Fish et al. (35) found that the Stokes radius of the SDS complex was similar to that of the protein in 6 M guanidinium chloride. This suggests gross disorganization.
294
PITTZ
AND BELL0
Solvent Perturbation Di$erence Spectra Herskovits and Laskowski (2) in studying solvent perturbation difference spectrophotometry of RNase in 150 mM SDS did not use SDS in the model system. They found A’sP/e~ to be 0.020 for RNase in 150 mM SDS using 20 % sucrose as perturbant, while we find Acsp/cM to be 0.016. (0.029 given by Herskovits and Laskowski for the perturbant sucrose in their Table III should be 0.020; T. T. Herskovits, personal communication.) They suggested that the exposure of tyrosyls of RNase in water and in 150 mM SDS is the same, about 50 %. An alternate explanation proposed by them was that SDS binds to tyrosyls, masking them from the perturbant, although no test was done. If there is no masking of tyrosyls by SDS, the conclusion of Herskovits and Laskowski that exposure is about 50% (44% from our data) in 150 mM SDS would probably be correct. If there is masking of tyrosyls by SDS, the procedure of Herskovits and Laskowski can provide no information on the number of external tyrosyls. On the hypothesis that tyrosyls of RNase and of appropriate models are partially masked, we calculate a variety of estimates of external tyrosyls for RNase in 150 m&r SDS. Glu-tyr-glu, which probably is not masked, gives the lowest estimate of external tyrosyls, similar to that of Herskovits and Laskowski based on acetyltyrosine ethyl ester without SDS. RCMRNase, which is expected to be most like RNase gives 5-6 external tyrosyls at lo-20% sucrose. From Table II we see that the SDS-model interactions decrease Aesp/eM by about 50%. Therefore, if 50 % of the tyrosyls _.^. are external, as in native RNase, and if they interact with SDS as do those of the positively charged models, the observed Acs,/cM (with 20% sucrose) would be about 0.01, not 0.02, indicating 50 % external tyrosyls with our model system, and 25% exposed tyrosyls with the model (no SDS) of Herskovits and Laskowski. The crucial point lies in the terms external and exposed. If all of the tyrosyls are external, but are, on the average, 50% exposed (i.e., 50% masked), we have concordance between the interpretations of Herskovits and Laskowski and of ourselves.
The linearity of the Hamaguchi-Kurono plots (37) (Fig. 4a) for RNase and the models in water indicates that access of the perturbant to the chromophore is independent of perturbant concentration. The upward deviation of AEJE~ for SDS and 20% sucrose does not necessarily indicate that 20 % sucrose causes some dissociation of SDS from the tyrosyl to give a AEJE~ close to that in the absence of SDS. Dissociation would decrease AEJQ because the shift of tyrosyl from hydrophobic medium to water would cause a blue shift greater than the red shift of solvent perturbation, unless there were a red shift arising from burial. The increased AQ,/Q at 20 % sucrose may mean that with increasing sucrose concentration there is relatively increased binding of sucrose and/or SDS. Gratzer and Beaven (38) found that sucrose slightly decreased, and ethylene glycol increased the critical micelle concentration of a nonionic detergent. A more dramatic effect of a cosolvent has been observed by us when 20 % ethanol or 2-methyl-2,4-pentanediol is added to RNase in 30 mM SDS: the 280-nm CD extremum is restored to that of native RNase. (This supports the idea that charges are less important than hydrophobic forces in the binding of SDS to RNase.) In the case of the solvent perturbation difference spectral results, we again face the problem of the appropriateness of the models, especially of RCM-RNase. In water RCMRNase appears to be well exposed to solvent since the solvent perturbation spectra of Herskovits and Laskowski (2, 39) and of ourselves indicate similar Aesp/eM for RCMRNase and for ATA. In water RCM-RNase is well unfolded (4042). That a residuum of regular structure remains is suggested by CD studies of Klee (43) on S-peptide. It is encouraging to note that two small models give results similar to those of RCM-RNase, and that AeD for ATA approaches that of RCM-RNase as the SDS concentration increases. In SDS, our conclusions are valid only if the tyrosyls of RCM-RNase and RNase are in substantially similar states. Two of the tyrosyls of RNase are adjacent to d&sulfides, a third is two residues away, and a fourth is three residues away. This situation does not exist in RCM-RNase
RNase AND TYROSYL MODELS IN SDS Titration data (26) suggest that the tyrosyls of RCM-RNase interact somewhat differently with SDS than do those of RNase; but this is a high pH result and may not apply at the pH of this work. We cannot be perfectly confident about the appropriateness of RCM-RNase as a model; but no model can be exactly like RNase. With lys-t,yr-lys and tyramine, we calculate smaller fractions of external tyrosyls in RNase from larger AesP/ean values. The far ultraviolet CD data show that SDS induces an apparently more regular conformation in RCM-RNase. Even if SDS promotes some increase in helical content, the effect on the tyrosines is unknown, and helix formation need not bury tyrosyls. Tyrosyls in an SDS-helical region might be external and masked by SDS rather than buried in the protein. Perhaps SDS acts as “glue” between hydrophobic residues. terhaps tyrosyls are both buried and masked in a proteinSDS complex. The normally helical regions of RNase do not contain tyrosine, and the sequences in which tyrosines are found are not conducive to helix formation. Tyr-25 is in the sequence ser-ser-ser-asn-tyr-cys; tyr-73 and tyr-76 in the sequence thr-asn-cys-tyr-glu-ser-tyr-serthr-metser; tyr-92 and tyr-97 in the seser-ser-lys-tyr-pro-asp-cys-ala-tyrquence lys-thr-t#hr; tyr-115 is in the sequence pro-t,yr-val-pro. Temperature Perturbatum Diffewnce Spectra The decreasing magnitude of Aetp for ATA with increasing SDS concentration supports the idea tha,t it is the chromophore-Hz0 interaction which generates Ae6tp, because Aq, decreases with increasing interaction between the chromophore and SDS. The ratios of the AetP values for RNase and RCM-RNase in SDS are close to unity. As, for RNase is constant in O-53 mM SDS. This may be explained as: (1) no change in the conformation of RNase; (2) disruption of the native conformation of RNase with equivalent burial in a new conformation; or (3) counterbalancing effects of externalization and masking. The first of these is ruled out by other data (Ae, , CD, and fluorescence). We cannot decide between the
295
second and third explanations from AQ, data alone. Explanation 2 disagrees with ,,except for the possibility of burial in a A~D new conformation with AeD # 0. Both 2 and 3 require gross disorganization of RNase by SDS and are incompatible with the idea of little or no change. Explanation (3) is consistent with Acsp because the interaction of the models with SDS decreases A%, by 50 %; if this represent,s 50 % masking from solvent, we expect a similar decrease in Ae,, . We find a 45 % decrease for RCM-RNase and 34 % for ATA. At higher SDS concentration the decrease for ATA would probably be greater. Thus, the constancy of Aetp for RNase is consistent with a doubling of external tyrosyls (from 3 to 6) with concomitant halving of Ae,, per tyrosyl through masking. For RNase in water Xt, is about 3 nm smaller than those of RCM-RNase and ATA. The latter two have similar ht, values and the ratio of their Actp values is 6.2, supporting the idea that the tyrosyls of RCMRNase are exposed in HzO. The difference in Xt, between RNase and the models in H20 may arise from difference in hydration or water structure caused by groups close to tyrosyl. This might be expected to affect Aetp . But the number of external tyrosyls for RNase in Hz0 calculated from AC,, is in agreement with X-ray results and solvent perturbation results. If water structure and hydration of tyrosyls are affected by nearby groups, solvent perturbation should also be affected. Comparison of AeD , Aesp, and ACQ We have indicated that all three types of difference spectra are compatible with the hypothesis of 6 external, masked tyrosyls. But there are other compatible hypotheses. In Table V we show the AC values calculated for all possible distributions of tyrosyls, considering only integral numbers, and omitting those with 4 or more buried tyrosyls which give Ae values far from experimental (e.g., all AeD are positive). The experimental values are also given in Table V. The calculated Ae values were obtained on the following bases. For A+:AeD of exposure = -900 (one-third of - 2700) and At, of masking one exposed tyrosyl is f300
296
PITTZ
AND
(the average of RCM-RNase, lys-tyr-lys, and tyramine in 150 mM SDS adjusted upward by 2 % to give a round number). For Acsp/eM, we used the average value for exposed tyrosyl in Hz0 from RCM-RNase and all three tripeptides, and for masked tyrosyl we used the average for RCM-RNase, lys-tyr-lys, and tyramine in 150 mM SDS. In both cases20 % sucrose was the perturbant. There is a difficulty in using AE,*/E~ , in that, Q for native protein includes contributions from phenylalanine (small), disulfides and the enhanced E of buried tyrosyls. Thus comparison of AesP/eM for RNase with those of models contains an error, usually not, important, from these sources. We have compromised on the use of r, = 9000 for RNase, about midway between Edfor RNase and RCM-RNase. (In Table II we have used the actual Ed, or more accurately the observed Asp/AM.) Therefore the experimental AcsP/eXin Table V for RNase differs slightly from that in Table II. For Aebpin Hz0 we used the average of RCM-RNase and ATA, and in 53 mM SDS, the value for RCM-RNase. Table V shows that for three cases only do all three difference spectral methods give agreement,, examples 12, 17, and 18; and 18 (6 external, masked tyrosyls) gives the best agreement with experiment. Even if AetPis set aside as a relatively unproved technique, we find agreement between AE, and AesPonly for 12, 17, and 18. The agreement between Aesp , AeD, and As, supports the validity of Ace . In Table V, we have not tried to refine the results by the use of fractional residues. Instead we have allowed a range of AEvalues sufficient to account for some fractional residue in each category. The use of Ae, = - 2700 for total exposure may really repre-
sent only 2.5 tyrosyls. This would change the results somewhat, without changing the conclusions. There are other difficulties, e.g., the meaning of AC, of f300 for masking in SDS compared with +500 in HTAC, and the question of the adequacy of the models. For these reasons the main use of Table V is to indicate those distributions which are grossly incompatible with experiment, and those which are in the range of interest. Table V demonstrates the importance of using several methods.
BELL0 TABLE
V
COMPARISON OF EXPERIMENTAL A.& FOR RNASB IN SDS WITHTHEORETICALUI'SFORDIFFERENT COMBINATIONS OF BURIED, MASKED, AND ExPOSED TYROSYLB
T ES-PI e no.
Number of tzdsyls
external
EXpoSd -
Calcuhtcd spectral data
L\cD
--
1 2 3 4 5 6 7 8 9 10 11 12* 13 14 15 16 17* 1f3* 19 20 21
Experimental 3 3 3 3 4 4 4 5 5 2 2 2 1 1 1 1 0 0 0 0
0 3 2 1 0 1 2 0 1 2 3 4 2 3 4 5 6 5 4 3
AE’S
3 0 1 2 2 1 0 1 0 2 1 0 3 2 1 0 0 1 2 3
$600
0.017 0.017 0.026 0.023 0.020 0.023 0.026 0.029 0.029 0.032 0.017 0.020 0.023 0.011
0 -600 - 1200 -900 -300 +300 $900
0.017 0.020 0.017; 0.014; 0.0111 0.008~
-1000 0 - 1800 -1800 -600 -900 -1500 -2100 - 1800 -2400 -300 -900 - 1500
0.014
-185 -166 -245 -225 -196 -8’90 -250 -280 -275 -305 -170 -200 -230 -115 -145 -176 -206 -180 -160 -120 -90
- a Italicized numbers are in agreement with experimental within about 20%. Examples with * are those for which AND , AcSp , and Aetp agree.
Further Comments on Masking
Above pH 12 RNase and RCM-RNase show irreversible spectral changes arising from reaction of hydroxide ion at the disulfide and carboxymethylcysteinyl groups. Conversion of cystine to cysteic acid inhibits this effect (45). In 30 mM hexadecyltrimethylammonium chloride RNase shows the irreversible spectral effect below pH 10 (26). The low pH for this reaction in the cationic detergent suggests that cationic groups are close to the hydrophobic cystine residues. Dyson and Noltmann (20) studied the exposure of chromophores of phosphoglucose isomerasein SDS, without SDS in the model systems. They also found that the tyrosyls of this enzyme are titrated at higher pH in SDS than in water (46). They consider masking by SDS, but, conclude that if the
RNaae
AND
TYROSYL
spectra are affected, the “conclusions would still be qualitatively correct, although the numerical values for the exposed residues might differ somewhat” (20). Our data suggest that the numerical values of external tyrosyls may differ greatly. In conclusion, we have presented results that can be explained consistently on the hypothesis of disruption of the conformation of RNase with externalization and masking of tyrosyl residues. It is clear that the usual models, ATA and N-acetyltyrosine esters, are not useful at low to moderate detergent concentrations. Masking, or association of the apolar tail of a detergent with chromophores has been justifiably applied to red shift’s at low concentrations of detergents [e.g., Refs. (21), (47), (48)]. Our point is that if the red shifts of appropriate model compounds are not considered, we cannot attempt to interpret blue shifts. A blue shift means exposure, but it is probable that this is usually accompanied by some degree of masking of both normally and newly external tyrosyls. At low detergent concentrations, at which a few detergent molecules are bound, it is probable that in many cases there is no externalization and the red shift represents masking only. But in some cases there may be externalization with a net red shift arising from a greater amount of masking than externalization. REFERENCES 1. BIGELOW, C. C., AND SONENBERG, M., Biochemistry 1, 196 (1962). 2. HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Sol. Chem. 243, 2123 (1968). 3. SELA, M., WHITE, F. H., JR., AND ANFINSEN, C. B., Science 136, 691 (1957). 4. SELA, M., WHITE, F. H., JR., AND ANFINSEN, C. B., Biochim. Biophys. Acta 31,417 (1959). 5. BELLO, J., Biochemistry 8, 4542 (1969). 6. BELLO, J., Biochemistry 8, 4550 (1969). 7. CANE, M., Fed. Proc. Fed. Amer. Sot. Exp. Biol. Abstr. 28, 2 (1969). 8. LASKOWSKI, M., JR., LEACH, S. J., AND SCHERAGA, H. A., J. Amer. Chem. Sot. 82, 571 (1960). 9. LEACH, S. J., AND SCHERAQA, H. A., J. Amer. Chem. Sot. 82, 4790 (1960). 10. HERSKOVITS, T. T., AND LASKOWSKI, M., JR., J. Biol. Chem. 237, 2481 (1962). 11. COWGILL, R. W., Biochim. Biophys. Acta 120, 196 (1966).
MODELS
IN
SDS
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12. COWGILL, R. W., Arch. Biochem. Biophys. 104, 84 (1964). 13. KUNITZ, M., J. Biol. Chem. 164, 563 (1946). 14. BIGELOW, C. C., J. Biol. Chem. 236, 1706 (1961). 15. SARFARE, P. S., AND BIGELOW, C. C., Can. J. Biochem. 46, 651 (1967). 16. SELA, M., AND ANFINSEN, C. B., Biochim. Biophys. Acta 24, 229 (1957). 17. SIMMONS, N. S., AND GLAZER, A. N., J. Amer. Chem. Sot. 89, 5040 (1967). 18. JIRGENSONS, B., AND CAPETILLO, S., Biochim. Biophys. Acta 214, 1 (1970). 19. RAY, A., REYNOLDS, J. A., POLET, H., AND STEINHARDT, J., Biochemistry 8, 2606 (1966). 20. DYSON, J. E. D., AND NOLTMANN, E. A., Biochemistry 8, 3544 (1969). J. A., HERBERT, S., AND 21. REYNOLDS, STEINHARDT, J., Biochemistry 7, 1357 (1968). 22. COWGILL, R. W., Biochim. Biophys. Acta 140, 37 (1967). 23. COWGILL, R. W., Biochim. Biophys. Acta 133, 7 (1967). 24. COWGILL, R. W., Arch. Biochem. Biophys. 100, 36 (1961). 25. BIGELOW, C. C., J. Theor. Biol. 16,187 (1967). 26. BELLO, J., Arch. Biochem. Biophys., submitted for publication. 27. SIMPSON, R. T., AND VALLEE, B. L., Biochemistry 6, 2531 (1966). SCATTURIN, A., AND 28. TAMBURRO, A. M., MORODER, L., Biochim. Biophys. Acta 164, 583 (1968). 29. PFLUMM, M. E., AND BEYCHOK, S., J. Biol. Chem. 244, 3973 (1969). 30. HORWITZ, J., STRICKLAND, E. H., AND BILLUPS, C., J. Amer. Chem. Sot. 92, 2119 (1970). M. E., AND 31. WOODY, R. W., FRIEDMBN, SCHERAGA, H. A., Biochemistry 6, 2034 (1966). 32. YANARI, S., AND BOVEY, F. A., J. Biol. Chem. 236, 10 (1960). 33. REYNOLDS, J. A., AND TANFORD, C., Proc. Nat. Acad. Sci. U.S.A. 66, 1002 (1970). 34. REYNOLDS, J. A., AND TANFORD, C., J. Biol. Chem. 246, 5161 (1970). 35. FISH, W. W., REYNOLDS, J. A., AND TANFORD: C., J. Biol. Chem. 246, 5166 (1970). 36. PITT-RIVERS, R., AND IMPIOMBATO, F. S. A., Biochem. J. 109, 825 (1968). 37. HAMAGUCHI, K., AND KURONO, A., J. Biochemistry Tokyo 64, 111 (1963). 38. GRATZER, W. B., AND BEAVEN, G. H., J. Phys. Chem. 73, 2270 (1969). 39. HERSKOVITS, T. T., AND L~SKOWSKI, M., JR., J. Biol. Chem. 236, 11 PC 56 (1960). 40. HARRINGTON, W. F., AND SELA, M., Biochim. Biophys. Acta 31, 427 (1959).
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41. HARRINGTON, W. F., AND SCHELLMAN, J. A., C. R. Trav. Lab. Carlsberg 50, 21 (1956). 42. KLEE, W. A., Biochemistry 6, 3736 (1967). 7, 2731 (1968). 43. KLEE, W. A., Biochemistry 44. TANFORD, C., J. Amer. Chem. Sot. 84, 4240 (1962). 45. TRAMER, Z., AND SHUGAR, D., Acta Biochim. Polon. 6, 235 (1959).
BELL0 46. DYSON, J.E.D., AND NOLTMANN, E. A., Biochemistry 8, 3533 (1969). 47. ZAKREZEWSKI,K., AND GOCH,H., Biochemistry 7, 1835 (1968). 48. STEINHARDT, J., AND REYNOLDS, J. A., “Multiple Equilibria
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Chap. 8. Aca-