titration of tyrosyl compounds in cationic, anionic and nonionic detergents

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 36190

TITRATION OF TYROSYL COMPOUNDS IN

CATIONIC, ANIONIC AND NONIONIC DETERGENTS

45

JAKE BELLO AND HELENE R. BELLO Department of Biophysics, RosweU Park Memorial Institute, Buffalo, N . Y . z 4 203 (U.S.A.)

(Received April 24th, 1972)

SUMMARY

Spectrophotometric titrations of the tyrosyls of ribonuclease, reduced ribonuclease and positively charged model compounds in sodium dodecyl sulfate result in changes in pK, sharpness of titration curves, spectral shifts and changes in absorbance. At high pH the interactions with sodium dodecyl sulfate are weak. Titration of ribonuclease and negatively charged models in the presence of the cationic detergent hexadecyltrimethylammonium chloride results in lower pK values, red shifts at both neutral and high pH, and high absorbances. These results are suggested to arise from binding of detergent to or near the phenolic groups.

INTRODUCTION

The interactions of detergents with proteins continues to be a subject of basic and utilitarian interest. In the presence of an anionic detergent increases in pK and changes in sharpness of tyrosyl titrations of proteins have been observed a-4. Several explanations of such data have been proposed. Zakrzewski and Goch 1 suggested that dodecanoate interacts with the "environment of the tyrosyl residues", but also suggested that conformational changes are the source of the titration effects. Lovrien s studied sodium dodecyl sulfate-albumin at low sodium dodecyl sulfate concentration. Considering it unlikely that the increase in tyrosyl pK arose from direct interaction with the phenolic group, he discussed the data in terms of conformational effects. In the case of complexes of serum albumin of dodecylbenzenesulfonate, Decker and Foster 2 found complex behavior of the pK values of several titratable groups. They considered overall electrostatic effects and conformational differences. Dyson and Noltmann 4 found that in the titration of a phosphoglucose isomerase without detergent, all of the tyrosyls are difficultly accessible and are t i trated (with time-dependent

Abbreviations: Brij-35 , a proprietary name for polyoxyethylene lauryl ether; HTAC, hexadecyltrimethylammonium chloride; RCAM-ribonuclease, ribonuclease with the disulfides reduced and earboxamidomethylated; TNBS, trinitrobenzenesulfonic acid.

Biochim. Biophys. Acta, 278 (1972) 45-56

46 J. BELLO, H. R. BELLO

spectral changes) at above-normal pH. In 1% sodium dodecyl sulfate the titration curve was at still higher pH. The sulthydryl groups could be ti trated in sodium dodecyl sulfate with pK lO.2. The explanation of Dyson and Noltmann 4 was that sodium dodecyl sulfate causes dissociation to subunits with exposure of sulihydryl, but that all of the tyrosyls remain unavailable. But since no time-dependence was reported for titration in sodium dodecyl sulfate, we may consider the alternative explanation, that in sodium dodecyl sulfate the tyrosyls are available, but that the pK is raised by the sodium dodecyl sulfate. The pK of lO.2 for sulihydryl is at the high end of the range for this group, and may arise from the effect of sodium dodecyl sulfate. The pK for the sulfhydryl of glutathione is 9.2, and pK values of about IO are observed when a negative charge is nearby 5. The effects of sodium dodecyl sulfate on sulfhydryl and tyrosyl may be similar, i.e. an increase in pK in both cases, arising from association of the titratable side chains with anionic detergent.

In an earlier paper 6 we presented spectral evidence for the partial masking of tyrosyls by detergents, the effect being strongly dependent on the presence of electric charges in the model compound. In this report we describe the titration of the phenolic groups of several tyrosyl derivatives in the presence of anionic, cationic and nonionic detergents. The results can be interpreted in terms of the binding of detergent to the chromophores.

MATERIALS AND METHODS

Ribonuclease A was Worthington phosphate-free. Ribonuclease with reduced and carboxamidomethylated disulfides (RCAM-ribonuclease) was prepared by reduction with dithiothreitol in 8 M urea at pH 8, followed by alkylation with bromo- acetamide, filtration through Sephadex G-25 and freeze-drying. Acetylation was done in 0.5 M sodium acetate with 0.2 ml acetic anhydride per IOO mg ribonuclease, followed by gel-filtration and freeze-drying. Guanidinated ribonuclease was prepared by treating 58 mg ribonuclease with 2 g guanyldimethylpyrazole nitrate in 5 ml of water at pH 9.5 for 22 h, followed by gel filtration and freeze-drying. Tyrosyl tripeptides were purchased from Schwarz/Mann Research Labs., and tyramine. HC1 from Calbio- chem. Sodium dodecyl sulfate (Sequanal grade) and polyoxyethylene lauryl ether (Brij-35) were obtained from Pierce Chemical Co., and hexadecyltrimethylammonium chloride (HTAC) from Eastman Organic Chemicals.

Analyses for amino groups were done by the trinitrobenzene sulfonic acid (TNBS) method of Habeeb 7. Titrations were carried out with KOH, and spectra were measured with a Cary Model 15 spectrophotometer at 27 °C.

Thermal perturbation spectra were done as described earlier s. Rates of iodination were measured spectrophotometricalty; at pH 7 by following the rate of disap- pearance of iodine at 400 nm; and at pH 11.5 by following the rate of formation of iodinated phenolic derivative at 305 nm.

RESULTS

p K effects In titrations with sodium dodecyl sulfate no corrections were made for the Na +

error, because the same concentration of NaC1 was present in the controls (although


TITRATION OF TYROSYLS IN DETERGENTS 47

the Na + activities were not the same in the two cases), and because the error would be the same throughout the series. Any lack of correspondence between solutions results in an error which is small relative to the significant effects observed. At the Na + concentration of o.o3 M the electrode error is o.o2 pH at pH I i (within the error of measurement) and 0.04 at pH II . 5. Above p H i I . 5 precipitation of sodium dodecyl sulfate-protein occurs. While the critical micelle concentration depends on ionic strength, the titrations were done above the critical miceUe concentration. I t has not been possible to complete some of the titrations in sodium dodecyl sulfate because of the afore-mentioned precipitation at high pH. An approximate pK can be estimated from extrapolation to the expected e at the end of the titration, although the final e cannot be predicted with certainty because the interaction of the tyrosyl with detergent varies with the tyrosyl compound. More accurate data above p H I I . 5 would not affect the conclusions.

I0 25

8 2 0

- ! J/J !:" r i i I I I

7 8 9 l0 II 12 ? 8 9 10 II 12

Fig. I . T i t ra t i ons w i t h and w i t h o u t 0.0 3 M sodium dodecyl sulfate. Le f t : C), r ibonucteaae; e , ribonuclease-sodium dodecy] sulfate D, guauidinated ribonuclease; I I , guamidinated ribonuclease- sodium dodecyl sulfate; ~ , acetylated ribonuclease; &, acetylated ribonuclease-sodium dodecyl sulfate; V, RCAM-ribonuclease-sodium dodecyl sulfate. Right: O, Lys--Tyr-Lys; O, Lys--Tyr- Lys with sodium dodecyl sulfate; A, Leu-Tyr-Leu; A, Leu-Tyr-Leu with sodium dodecyl sulfate; [], Glu-Tyr--Glu; B, Glu-Tyr-Glu with sodium dodecyl sulfate.

Many of the titration curves are displayed in Figs I and 2, and pertinent data and derived quantities in Table I. Some curves not shown in the figures are adequately represented in Table I. Sodium dodecyl sulfate and NaC1 concentrations are o.o3 M unless otherwise indicated.

The pK in the presence of sodium dodecyl sulfate (pKsDs) of ribonuclease is shifted above pK 0 by more than z unit. The pKs~s values of the model compounds, relative to pK 0, are related to their charges. Glu-Tyr-Glu and Leu-Tyr-Leu are substantially unaffected by sodium dodecyl sulfate. The largest changes are for positively-charged tyramine and Lys-Tyr-Lys . Tyramine in o.I5 M NaC1 showed time-dependent absorbances over much of the curve; e~ss am (phenolate form) de-

Biochim. Biophys. Acta, z78 (x972) 45-56

4 8 j . BELLO, H. R. BELLO

16

14

1 2 F d Q , -13o

I0 ' 0

x

~u 8

<.-

25

20 n~ x

15 r~

41- I ~ .d I I I - I 1 0

2 1 - l e e " / / A l l / -45

7 8 9 I0 II 12 7 8 9 10 II 12 pH

Fig. 2. T i t r a t i o n s in Bri j-35 and HTAC. Lef t side Bri j-35; O, r ibonuc lease in NaC1; ~ , r ibonu- clease in 1% Br i j ; II, r ibonuc lease in 4% Br i j ; O, G l u - T y r - G l u in 4% Br i j ; &, L y s - T y r - L y s in 4% Brij . R i g h t side in o.o 3 M H T A C : (D, r ibonue lease ; O, RCAM-r ibonuc lease ; I , G l u - T y r - G l u ; A, L y s - T y r - L y s .

T A B L E I

T I T R A T I O N D A T A F O R T Y R O S Y L C O M P O U N D S I N D E T E R G E N T S

Solute Solvent p K ApH a $maz gma* Aea/ZJeo b Neutral High pH

Ribonuc lease 0.03 M NaC1 I O . I c o.8oC 278 __d __ RCAM-r ibonuc lease 0.03 M NaC1 io .o 0.65 274 293 - - Ace ty l a t ed - r i bonuc l ea se 0.03 M NaC1 io.4e o.8o e 277 __d __ G u a n i d i n a t e d r ibonuc lease o.o 3 M NaCI 9.9 e o.65 e 277 __d __ L y s - T y r - L y s o.o 3 M NaC1 9.7 0.60 274 293 - - G l u - T y r - G l u o.03 M NaCI lO.2 0.57 274 292 - - L e u - T y r - L e u o.o 3 M NaC1 IO.O o.6o 274 292 - - p - H y d r o x y p h e n y l a c e t i c

acid 0.03 M NaC1 io .o 0.58 275 293 - - T y r a m i n e o.15 M NaC1 9-5 o.6o 274 293 - - R ibonuc lease 0.o3 M sod ium

dodecyl su l fa te (i 1.3)e __t 277 r __t RCAM-r ibonuc lease o.o 3 M sod ium

dodecy l su l fa te lO.6 0.40 276 293 1.o6 Ace ty l a t ed - r i bonuc l ea se 0.03 M sod ium

dodecy l su l fa te ( i i . 5 ) e __t 276 __t __t G u a n i d i n a t e d r ibonuc lease o.o 3 M sod ium

dodecy l su l fa te ( i i . 2 ) e (o.5)e 277 __f __t L y s - T y r - L y s 0.03 M sod ium

dodecyl su l fa te lO. 5 0.57 276 293 I . I I G l u - T y r - G l u o.o 3 M sod ium

dodecy l su l fa te lO.2 0.56 274 292 1.o2


TITRATION OF TYROSYLS IN DETERGENTS

TABLE I (continued)

49

Solute Solvent pK ApHa 2max 2max Aea/Aeo b Neutral High pH

Leu-Tyr -Leu 0.03 M sodium dodecyl sulfate 9-9 0.60 275 292 I.O2

Tyramine o.15 M sodium dodecyl sulfate lO. 7 o.35 276 293 1.o8

Ribonuclease o.i 5 M sodium dodecyl sulfate .(I°-9)e (o.7)e 276 t __a

Ribonuclease 1% Brij-35 e io.o o.95 e 277 __d I.O Ribonuclease 4% Brij-35 e io. 3 I.Oe 278 __d Lys--Tyr-Lys 4~/o Brij-35 e 9.6 0.70 275 292 0.97 Glu-Tyr-Glu r% Brij-35 lO.2 0.60 274 292 1.o2 Glu-Tyr-Glu 4% Brij '35 IO. 3 0.63 274 293 1.o2 Leu-Tyr-Leu 4% Brij-35 io.o 0.62 274 292 1.o 4 Ribonuclease

(first step) 0.03 M HTAC (9.3)h (o.6)n 278 __h __2a Ribonuclease

(second step) 0.03 M HTAC (xo.3)h (o.4)h __h 297 - -~ RCAM-ribonuelease 0.03 M HTAC 9.3 0.8 275 296 r . I6 Lys -Tyr -Lys 0.03 M HTAC 9.8 0.75 274 297 1.o6 Glu-Tyr--Glu 0.03 M HTAC 9-7 0.70 276 298 1.16 p-Hydroxyphenylacet ic

acid 0.03 M HTAC 9.5 0.70 276 298 I . i i Ribonuclease 8 M Urea t lO. 5 0.60 276 295 - - Ribonuclease 8 M Urea-

0.03 M sodium dodecyl sulfate lO.8 0.30 276 295 1.o4

a pH range of middle third of t i tration. b Ratio of Ae in detergent to As in NaC1, at high pH. e For first step of t i t ra t ion curve. a Tyrosinate and tyrosine bands overlap for first step of t i t ra t ion curve. e Est imated from incomplete t i t ra t ion curve.

Not known because of precipitation above pH 11.6. g Uncerta in because 4 tyrosyls are t i t ra ted in the 4~o Brij-35 and 3 in NaC1. h Uncerta in because of indistinct end or beginning, and t ime dependence of absorbance. i 0.03 M NaC1 present.

creased, while e~76 nm (unionized form) did not change. No pH change accompanied this effect. In o.15 M sodium dodecyl sulfate, there was a small time-dependent increase in the absorbance of tyramine at pH 9.5-io, at 293 nm, but no change at 276 nm. The tyramine data shown are from absorbances extrapolated to zero time. Tyramine, and p-hydroxyphenylacetic acid in the case of the cationic detergent, HTAC, were selected as models for which conformational effects would be unimpor- tant.

To test the importance of binding to positive charges in protein we acetylated ribonuclease on 90°//0 of the lysines. Above the titration range of the imidazole groups, of the original 15 positive charges 5 remain (4 guanidinium and I ammonium). The t i t r a t i o n c u r v e o f a c e t y l a t e d r i b o n u c l e a s e in 0 .03 M NaC1 is s h i f t e d t o h i g h e r p K t h a n

t h a t o f t h e 3 e x p o s e d t y r o s y l s o f r i b o n u c l e a s e . T h e t i t r a t i o n c u r v e o f a c e t y l a t e d r i b o -

n u c l e a s e in NaC1 a p p e a r s to h a v e a s t e p a t t h e e n d o f t i t r a t i o n o f 3 t y r o s i n e s , b u t t h i s

is m u c h less d i s t i n c t t h a n for n a t i v e r i b o n u c l e a s e , p r o b a b l y b e c a u s e t h e t i t r a t i o n o f

t h e f i r s t 3 t y r o s y l s o f a c e t y l a t e d r i b o n u c l e a s e is a t h i g h e r p H . T h e t i t r a t i o n c u r v e o f

a c e t y l a t e d r i b o n u c l e a s e in s o d i u m d o d e c y l s u l f a t e w a s s h i f t e d to h i g h e r p H , b u t n o t


5 ° j. BELLO, H. R. BELLO

quite as much as for ribonuclease. I t is difficult to make a quanti tat ive s tatement because complete titration curves could not be obtained. We guanidinated the lysines (to the extent of 90% as shown by TNBS) to convert them to groups not t i tratable in the normal tyrosyl range. The ti tration curve in o.03 M NaC1 (Fig. I) shows that ap- proximately 4 tyrosyls can be t i t rated readily with an average of p K o 9.9, but the 4 may not all have the same pK 0. This was not studied with sufficient precision to de- cide. Klee and Richards 9 found 3 readily t i tratable tyrosyls for more fully guanidinated ribonuclease. In sodium dodecyl sulfate the ti tration curve of guanidinated ribonuclease shifted toward higher pH than in o.03 M NaC1. The shift for guanidinated ribonuclease appears to be somewhat greater than for ribonuclease. The data for acetylated ribonuclease and guanidinated ribonuclease indicate some contribution from cationic groups toward binding of sodium dodecyl sulfate, but not a dominant role.

For ribonuclease in I % Brij-35, the ti tration curve was similar to that in water, with 3 tyrosyls being t i t rated with p K IO.O, and the remainder in a second wave above pH 11. 7. In 4% Brij-35, 4 tyrosyls were t i t rated with an average p K of lO.3. The pK values for the tripeptides in 4% Brij-35 were similar to the p K 0 values. All of the Brij-35 solutions contained 0.03 M NaC1.

With the cationic detergent, HTAC, negative charge was important . For Glu- Tyr -Glu and p-hydroxyphenylacetic acid HTAC lowered the pK by 0. 5, but there was no decrease for Lys -Tyr -Lys . For ribonuclease from pH 9.7 and higher, a time-dependent increase in absorbance was observed, slow at pH 9.7, and becoming faster with increasing pH. The data shown are for zero time. (In order to measure the spectra close to zero time, KOH was added to the solution in the cuvette, the spectrum was immediately taken, and the pH was measured after the spectrum was taken. Control experiments, in which the pH was also measured before the spectra, showed no change during the interval.) The time-dependent effects appear to be a combination of those seen in 8 M urea containing alkali sulfates 1°, which are explicable as unfolding of ribonuclease, and those seen above pH 12 in water, and at tr ibuted to reactions at disulfides 1°-14. With increasing pH the latter effect dominates. The two effects are easily distinguished, the reaction at disulfides resulting in a general irreversible rise in absorbance, while the unfolding results in a specific reversible rise at 295 rim. With oxidized glutathione (disulfide form) time dependence was seen over the wavelength range studied, 350-25 ° n m , with the rate of increase of absorbance at p H I I five times larger in HTAC than in water. For oxidized ribonuclease (cystine oxidized to cysteic acid) in HTAC, the rate of absorbance change was less than 5 % of that of ribonuclease. Since high pH reactions at the cystines and carboxamidomethylcysteinyls require a t tack by OH-, the facilitation of the reaction in the presence of HTAC suggests that H-TAC binds to the cystine regions to produce a positively charged environment.

The ti tration of ribonuclease in HTAC shows indication of a plateau near pH IO; this is indistinct because the time-dependent second step overlaps the first. The p K values for the two steps are about 9.3 and lO.3. Thus, t i tration of the normal tyrosyls and the release of the buried tyrosyls take place at lower pH in HTAC than in NaC1. The p K of RCAM-ribonuclease in HTAC is 9.3. Time dependence was observed from p H IO upward. In this case the time-dependent effect indicated only the irreversible reaction at the carboxamidomethylcysteinyl residues. In water RCAM-ribonuclease


TITRATION OF TYROSYLS IN DETERGENTS 5 I

shows time-dependent spectral changes above pH 12. There was no time dependence in the titrations of the small models in HTAC.

Widths of titration curves Table I shows the widths of the titration curves as ApH for the middle 0.33 of

the curve. The theoreticalApH is 2 log 2, or 0.60. In water ApH for the tripeptides and RCAM-ribonuclease are close to theoretical, but for ribonuclease ApH is 0.8 for the first step. In 0.03 M sodium dodecyl sulfate, the tripeptides show near-theoretical ApH values. ApH for ribonuclease and acetylated ribonuclease in sodium dodecyl sulfate are not known because of precipitation at high pH, but the curves are sharpened. For guanidinated ribonuclease in 0.03 M sodium dodecyl sulfate a crude estimate of 0. 5 has been made based on an extrapolated final e of 15 • lO 3 for titration of 6 tyrosyls. The curve is steeper than normal. ApH in sodium dodecyl sulfate is less than in water, as the curve is steeper for sodium dodecyl sulfate. For RCAM-ribonuclease in sodium dodecyl sulfate ApH is the relatively low value of 0. 4. ApH for tyramine has the low value of 0.35, but for Lys-Tyr -Lys ApH is normal.

In HTAC the model tripeptides and RCAM-ribonuclease show ApH values above theory, even for Lys-Tyr-Lys , for which there is no decrease in pK. Extrapola- tion of the first step for ribonuclease in HTAC to completion gives an estimated ApH of about 0.6; and extrapolation of the second step to its beginning gives a small ApH of about 0. 4 .

In 4°/0 Brij-35 the tripeptides show ApH values near theory, but with ApH for Lys-Tyr -Lys somewhat high at 0. 7. A pH for four tyrosyls of ribonuclease in 4% Brij- 35 is high at I.O. (The titrations in 4% Brij-35 with and without 0.03 M NaC1 were identical.) In i% Brij-35, ApH for ribonuclease was 0.95 for the first step, greater than in water, while the pK was io.o, nearly the same as in water. Thus even when detergent does not affect pK, it can affect ApH.

A, for tyrosine-tyrosinate Further information on tyrosyl-detergent interactions may be obtained from

Ae between the bottom and top of the titration curve. Since Ae is sensitive to the presence of spectrally inert impurities, we show the ratio of Aed in detergent to Ae 0 in NaC1. For sodium dodecyl sulfate, A ea/A e o for the tripeptides is in accord with the pK changes, being largest for those peptides with the largest pK changes. For the cationic detergent HTAC A ea/A eo values for Glu-Tyr-Glu and p-hydroxyphenylacetic acid are large, in agreement with ApK and ApH. Aeo/A, o for Lys-Tyr-Lys , while smaller than for Glu-Tyr-Glu, indicates an interaction with HTAC.

For Brij-35, Aeo/Aeo is close to unity for all 3 tripeptides. Ribonuclease in 1% Brij-35 shows A ed/A eo ofo.95 (using the ill-defined A e 0 for the first step of ribonuclease in water. For ribonuclease in 4% Brij-35 we cannot confidently present a value of Aea/Ae o, because the top of the first step is indistinct.

Wavelength of absorption band; interactions at high pH Another indicator of interaction is the wavelength of the tyrosyt or tyrosinate

absorption band. At neutral pH sodium dodecyl sulfate shifts 2max to the red for Lys- Tyr -Lys and tyramine, but in sodium dodecyl sulfate the tyrosinate forms of all the compounds have 2max at 292-293 nm, as in NaC1. Going from NaC1 to Brij-35, we see

Bioehim. Biophys. Aaa, 278 (i972) 45-56


little effect on 2max at either neutral or high pH. In HTAC we see a marked effect on 2max of the tyrosinate form of all compounds studied, even for L y s - T y r - L y s for which p K is little affected. 2max at neutral pH was also affected for the negative models.

The normal 2max of 292-293 nm for tyrosinate in sodium dodecyl sulfate suggests that interaction does not occur between negative tyrosinate and negative dodecyl sulfate. This was studied by two other methods, iodination and thermal perturbation difference spectrophotometry. Iodinations were done on tyramine andp-hydroxyphenylacetic acid at pH 5.85 and 11. 5 with and without 0.03 M sodium dodecyl sulfate. p-Hydroxyphenylacet ic acid was selected as a model which does not interact, or interacts very weakly, with sodium dodecyl sulfate. Iodination results are shown in Table II . They show that the rate of iodination of tyramine in sodium dodecyl sulfate

T A B L E II

IODINATION OF TYRAMINE AND p - H Y D R O X Y P H E N Y L A C E T I C ACID

Substrate p H Rate without sodium dodecyl sulfate relative to rate upith sodium dodecyl sulfate (o.o3 M)

Tyra rn ine 5.85 4.o 11. 5 1.6

p - H y d r o x y p h e n y l a c e t i c acid 5.85 i . i 11. 5 i . I

is decreased significantly more at pH 5.85 than at pH 11.5, but that even at pH 11. 5 the rate in sodium dodecyl sulfate relative to that without sodium dodecyl sulfate is not as small as for p-hydroxyphenylacetic acid. This indicates a reduced, but not a total elimination of, tyramine-sodium dodecyl sulfate interaction at pH 11.5. The moderate reduction of the rate of iodination of tyramine in sodium dodecyl sulfate is in accord with earlier data e which indicated that sodium dodecyl sulfate covers, but not completely, the phenolic groups of models and proteins.

The thermal perturbation spectra of tyramine with or without sodium dodecyl sulfate, and of p-hydroxyphenylacetic acid with or without HTAC, all near pH 11. 4, are shown in Fig. 3. The spectrum for p-hydroxyphenylacetic acid in HTAC is mar- kedly different from that obtained without HTAC, in accord with the difference in ,~max at high pH. But the spectra for tyramine with and without sodium dodecyl sulfate differ much less. We may note that the negative ext remum at long wavelength is shifted I nm to the blue by anionic sodium dodecyl sulfate and 6 nm to the red by HTAC. ~max (Table I), iodination and thermal perturbation spectra all show that at high pH the interaction of tyramine with sodium dodecyl sulfate is less than it is near neutral pH.

Titration in 8 M urea In 8 M urea, o.o3 M sodium dodecyl sulfate raises the p K of ribonuclease. The

ApH values were 0.6 in 8 M urea and 0.3 in 8 M urea-o.o3 M sodium dodecyl sulfate. The latter is the smallest ApH we have observed. The pH ranges for 75% and 90% of the t i tration curve were also about half of theory for ribonuclease in urea-sodium dodecyl sulfate. The ratio of Aeo for ribonuclease in 8 M urea-sodium dodecyl sulfate


TITRATION OF TYROSYLS IN DETERGENTS 5~

p - HYDROXYPHIENYLACETIC ~ lO pH 11.42

0.1 . ~ " ~ pH 11.45

.,Jr \ sos,f ~=~.'~ NO SDS

I I I I I I I I ;~ 0 290 310 330 350

x,nm Fig. 3. Thermal per turbat ion spectra of tyramine and p-hydroxyphenylaeetic acid with and without detergent. Temperatures are 4.7 °C in the "sample" and 26. 7 °C in the "reference" compart- ments of the Cary-I 5 spectrophotometer. Concentration of chromophores is 0.070 mg/ml. SDS = sodium dodecyl sulfate.

for ribonuclease in 8 M urea--o.o3 M NaC1 is slightly greater than unity. This is to Ae0 of interest in view of the effect of urea in inhibiting h~drophobic aggregation15, x6. But the 2 values are not affected by sodium dodecyl sulfate. Zakrzewski and Goch 1 have observed binding of dodecanoate ion to human serum albumin in 8 M urea.

DISCUSSION

The effects of the detergents on the tyrosyl compounds show complex interactions. The data for the tripeptides and tyramine indicate a dominating role of charge for the binding of sodium dodecyl sulfate. The yet higher pK for ribonuclease in sodium dodecyl sulfate compared with Lys-Tyr-Lys, and the results for acetylated ribonuclease, suggest important contributions from other interactions, probably in- cluding binding to hydrophobic groups*, 17-19.

Sodium dodecyl sulfate The pK of RCAM-ribonuclease in sodium dodecyl sulfate is lower than that of

ribonuclease, and is near those of the cationic models. (But for HTAC there is no difference between RCAM-ribonuclease and the first step of ribonuclease.) Presumably the higher pK of ribonuclease in sodium dodecyl sulfate arises from the conformational constraint imposed by the disulfides and the fact that all of the tyrosyls are near disulfides. This might result in sodium dodecyl sulfate producing a higher negative charge density in the disulfide--tyrosyl regions of ribonuclease than of RCAM-ribonuclease, despite the fact that the latter binds more sodium dodecyl sulfate overall than does the former s°. I t is general that non-disulfide proteins bind more sodium dodecyl sulfate than do disulfide-containing proteins "-s2. Nevertheless, it is not in- conceivable that ribonuclease binds more sodium dodecyl sulfate to its disulfide regions, in which the tyrosyls are located, than RCAM-ribonuclease does to its corre- sponding regions. We do not yet have information on the density of binding of detergents to specific regions of proteins. Or, if there is not more binding, the bound sodium dodecyl sulfate ions may be differently arranged on these regions in the two cases, leading to the difference in pK. Zakrzewski and Goch 1 have suggested that the disulfide regions of serum albumin are important for the binding of alkanoates.

Biockim. Biophys. Aaa, 278 (x972) 45-56


The red shifts of the spectra of tyramine and Lys-Tyr-Lys at neutral pH also point to interactions with sodium dodecyl sulfate. ~max at neutral pH was 276-277 nm for ribonuclease, RCAM-ribonuclease and the two positively-charged models in sodium dodecyl sulfate. For ribonuclease this represents an overall blue shift, which is probably the resultant of a larger blue shift of unfolding and a red shift of association with sodium dodecyl sulfate. This has been discussed in detail elsewhere 6, and here we shall note only that in sodium dodecyl sulfate 2m~x of ribonuclease at neutral pH is more like those of the positive models than that of Glu-Tyr-Glu.

The results for the simplest model, tyramine, are consistent with the idea that sodium dodecyl sulfate interacts with the tyrosyl chromophore. The increase in pK may arise from masking of the tyrosyl by sodium dodecyl sulfate and/or from the presence of negative sulfate groups close to, perhaps hydrogen bonded to, the hydroxyl. Partial burial of tyrosyls within a protein may also raise the pK, either by putting the hydroxyls close to negative charges or by formation of hydrogen bonds with appro- priate groups. For tyramine this is not possible.

2max at high pH in sodium dodecyl sulfate is the same as without sodium dodecyl sulfate. Repulsion between phenolate and anionic sodium dodecyl sulfate would bring the ionized hydroxyl into water with a shift toward normal )Lmax. We should not ex- pect that repulsion between phenolate an d dodecyl sulfate would necessarily dissociate the hydrophobic portion of sodium dodecyl sulfate from the phenol ring. But it appears to be the case that in the absence of a charge opposite to that of the detergent ion there is little, if any, interaction. This is clearly shown by the weak interaction of sodium dodecyl sulfate with N-acetyl tyrosine amide compared with tyramine 8, and by similar examples in the tryptophan series, still to be published. The iodination data and thermal perturbation spectra indicate a marked decrease, but not a total aboli- tion, of interaction with sodium dodecyl sulfate at high pH. Since these data and z]ed/Ae0 appear to disagree with 2max at high pH, further work is required in order to understand the discrepancy.

With regard to the widths of the titration curves for sodium dodecyl sulfate, those of the tripeptides are near 0.60, but those of RCAM-ribonuclease and oftyramine are much sharper. Small z]pH values in sodium dodecyl sulfate may be related to titration of ammonium groups with resultant changes in binding of sodium dodecyl sulfate. This would not appear to explain the difference between tyramine and Lys- Tyr-Lys.

The small A pH for RCAM-ribonuclease may arise from a cooperative conformational change, perhaps induced by the ionization of tyrosyl hydroxyls and deprotona- tion of ammonium groups. The extremely sharp titration curve for ribonuclease in urea-sodium dodecyl sulfate might be the result of an especially cooperative disrup- tion of conformation arising from the addition of the disruptive effect of urea to that of high pH. Conformational change cannot account for the small A pH for tyramine. In this case there is the possibility of aggregation to be considered. The nature of these changes lies outside the scope of this report and will be the subject of future work.

H T A C The cationic detergent HTAC lowers the pK of Glu-Tyr-Glu, in accord with the

spectral observations e on the interaction of HTAC with this peptide. For ribonuclease in 0.03 M HTAC, the time-dependent absorbance changes and the step near pH IO

Biochim. Biophys. Acta, 278 (I972) 45-56

TITRATION OF TYROSYLS IN DETERGENTS ~5

show that the buried tyrosyls become exposed at lower pH than in water. The near absence of effect of HTAC on the pK of Lys-Tyr-Lys suggests that the positive charges of this peptide inhibit binding of the cationic detergent. As the positive ammonium groups are discharged at higher pH we find binding of HTAC, as indicated by 2m~x of the tyrosinate form, which is 297 nm, almost as high as for Glu-Tyr-Glu. A ea/A eo for Lys-Tyr-Lys in HTAC is significantly above unity although less than for ribonuclease and Glu-Tyr-Glu.

A pH for RCAM-ribonuclease in HTAC is larger than in water; in this case titration of the ammonium group will not decrease HTAC binding, but increase it by increasing the net negative charge. However, the net effect of removing some positive charges and adding others is not obvious. Conformational studies are anticipated on peptides in HTAC, as a function of pH. The small ApH (o.4) for the second step of ribonuclease in HTAC probably arises from the unfolding of the protein indicated by the time dependence. The meaning of the 0.6 value of ApH for the first step of ribonuclease in HTAC is obscured by the time dependent effects noted in Results.

With ribonuclease, 4% Brii-35 causes a small increase in pK and some opening of the structure with indication of titration of 4 tyrosyls in the first step instead of 3. The large values of ApH for ribonuclease in 4% Brij:-35 indicates an interaction.

GENERAL CONSIDERATIONS

The effects of detergents on the tripeptides, on ribonuclease and on RCAM- ribonuclease might arise from folding of the molecule to bury the tyrosyl. In the cases of tyramine and p-hydroxyphenylacetic acid this cannot occur. For these compounds the most reasonable explanation is that detergent is closely associated with the chromophore and the hydroxyl groups.

Although not all aspects of the data are understood, the results of this work show that binding of detergents to tyrosyl model compounds gives titration effects similar to those observed with proteins. While some authors have recognized this possibility, it has not been generally accepted, resulting in explanations emphasizing effects other than detergent-tyrosyl interactions. At the detergent concentrations we have used, proteins do undergo drastic conformational and dissociative effects. Never- theless, titration data in such cases have sometimes been interpreted in such terms only, without taking into account interactions between titratable groups and detergents. The data also show that no one parameter of the titration curve can be relied on as an infallible indicator of detergent-tyrosyl interactions, but that several must be considered and that auxiliary data (such as the thermal perturbation spectra and iodination we have used) are helpful.

The results for ribonuclease in HTAC and Brij-35 suggest the possibility of partial unfolding for selective chemical modification of proteins. The use of ionic detergents to facilitate or inhibit chemical modification of proteins by charged reagents, e.g. reduction of disulfides, might be useful. This type of effect was first reported by Steinhardt and Fugitt ~ for acid hydrolysis of proteins.

Biockira. Biophys. Acta, 278 (i97 z) 45-56


ACKNOWLEDGEMENTS

Supported, in part by Grants GM 14oo 4 and GM 13485 from the Institute of General Medical Sciences, National Institutes of Health, and Grant GB 20083 from the National Science Foundation.

R E F E R E N C E S

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titration of tyrosyl compounds in cationic, anionic and nonionic detergents

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