Nuclear magnetic resonance study of the interaction of sodium dodecyl sulfate with phycocyanin

BIOCHIMICA ET BIOPHYSICA ACTA BBA

31

35306

N U C L E A R MAGNETIC RESONANCE STUDY OF T H E I N T E R A C T I O N OF SODIUM D O D E C Y L S U L F A T E W I T H P H Y C O C Y A N I N

R. M. R O S E N B E R G * , H. L. C R E S P I AND J. J. K A T Z

Chemistm, Division, Argonne National Laboratory, Argonne, Ill. 60439 (U.S.A.) (Received J u l y 22nd, 1968)

SUMMARY

The interaction of sodium dodecyl sulfate with the protein C-phycocyanin was investigated by proton magnetic resonance spectroscopy. Fully deuterated phycocyanin is used to make observation of the surfactant resonance possible without interference by resonance peaks of methyl and methylene groups on the protein. At low ratios of surfactant to deuterio-phycocyanin, the resonance peaks of the surfactant are greatly broadened, indicating a tightening of the protein structure as a result of the interaction. At higher ratios of surfactant to protein, all the resonance peaks of the surfactant are shifted upfield and moderately broadened, indicating that the entire hydrocarbon chain of the surfactant interacts with the protein, and that the surfactant is in a generally more hydrophobic environment when bound to the protein. Whereas interaction of small amounts of surfactant tighten the structure of the protein, high concentrations of sodium dodecyl sulfate appear to loosen the protein structure. The PMR spectra can be correlated with changes in the visible and ultraviolet spectra of deuterio-phycocyanin.

INTRODUCTION

The interaction between proteins and anionic surfactants has been of interest since 1935 when A~SON 1 showed that a commercial detergent (largely sodium dodecyl sulfate) denatures proteins at a lower concentration than do urea or gnanidine. The early electrophoretic studies of PUTNAM AND NEURATH2 led to the suggestion that anionic surfactants are bound to proteins through ion-pair formation with cationic sites on the protein, with the hydrocarbon portion of the surfactant free. They also postulated that a second layer of surfactant molecules was bound by hydrophobic interaction with the hydrocarbon chains of the first layer of surfactant. More recent studies by equilibrium dialysis ~-5, electrophoresis, 6-9 optical rotationS, 1°-14, difference spectroscopy 4,15, viscosimetry 5,n,16, volume change measurements 17, acid-base titration 16, and fluorescence measurements ~6have indicated that in fact the hydrocarbon chains of anionic surfactants are involved in the binding to protein, and that the bind* ACM R e s i d e n t F a c u l t y , 1967-68. P e r m a n e n t a d d r e s s ; C h e m i s t r y D e p a r t m e n t , L a w r e n c e U n i v e r s i t y , Appleton, V~:isconsin 54911. Biochim. Biophys. Acta, 175 (1969) 31 4 °

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ing of anionic surfactants produces conformational changes in the protein molecule. Observation of the proton magnetic resonance (PMR) spectrum ~,t a bound anionic surfactant should provide direct evidence for the involvelnent of the hydrocarbon portion of the surfactant ion in the interaction with the protein, and could provide additional information about the nature of the interaction and of the conformation changes produced. Unfortunately, the PMR peaks of the methyl and methylene groups of sodium dodecyl sulfate are in the same region of the spectrum as the corresponding groups in aliphatic side chains of the amino acids in the protein. Because of tile availability in our laboratory of fully deuterated algae grown in a medium containing 99.8 o4 "HoO (ref. 18), it becomes practical to use a fully deuterated protein to avoid this interference. We have therefore used completely deuterated C-phycocyanin l'a, a photosynthetic protein pigment, isolated from the fully, deuterated bluegreen alga Phormidium luridum. The presence of the tetrapyrrole chromot)hore, phycocyanobilin, whose absorption spectrmn is sensitive to protein conformation, provides an additional convenient means of monitoring conformational changes '°. Our experiments show clearly that the hydrophobic chain of dodecyl sulfate interacts with the protein, and that at low surfactant concentration the protein has a more rigid conformation than in the native state. High concentrations of dodecyl sulfate, however, produce increased motional freedom in the protein molecule*. EXPERIMENTAL

Materials The protein used was C-phycocyanin isolated from Phormidiu.m luridum grown in 99.8°,/0 2H20 nutrient medium 18. The fully deuterated C-phycocyanin used in this work is hereafter referred to simply as phycocyanin. A sample of 5o g (fresh wt.) of frozen cells was lysed in 25o ml o.oi M phosphate buffer (pH 6.9) by the addition of Io mg oflysozyme (Worthington, 2 × crystallized, ref. 19). After 2 h at 3°0 with stirring the material was stored overnight at 6 °. Then the suspension was centrifuged for IO rain at r 5 ooo x g in a Sorvall RC2-B refrigerated centrifuge at 6 °. The extraction was repeated one or more additional times to obtain the maximum yield of phycocyanin. The combined extracts were brought to 25/o o/ satn. of (NH4)2SO 4 by addition of saturated (NH4)~SO 4 solution 21. (All solutions used in the preparation were in H,,O except the final dialysate.) The resulting precipitate was centrifuged, washed with 25% saturated (NH4)2SO 4, and the combined supernatant fluid and washing was brought to 45 % saturation of (NH4)2SO a by addition of saturated (NH4)2SO 4 solution. The precipitated phycocyanin was centrifuged, and the pale blue supernatant fluid was discarded. The precipitate was washed with 45 (}o saturated (N Ha) 2SO4, tile washing discarded, and the washed precipitate was dissolved in IOO nfl of o.oi M phosphate (pH 6.9). The precipitation at 25 % saturated (NH4)2SO4 and 45 % saturated (N H4)~SO4 was then repeated. The final precipitate was dissolved in a minimum vol. of o.oi M phosphate buffer (pH 6.9) and dialyzed at 6 ° against three or more changes of buffer (io fold the w)lume of the protein solution). The purity of the preparation was judged from the ratio of the peak absorbance near 62o m# to the peak absorbance near 28o m/z in a solution whose absorbance at * A p r e l i m i n a r y r e p o r t of t h e s e r e s u l t s w a s p r e s e n t e d b e f o r e t h e l ) i v i s i o n of l ' h v s i c a l C h e m i s t r y , A m e r i c a n C h e m i c a l S o c i e t y , S a n F r a n c i s c o , A p r i l , I9()8.

Biochim. Biopl(~,s. Acta, 175 (i969) 3 i 4 °

SODIUM DODECYL SULFATE--PHYCOCYANIN INTERACTION

33

62o m# was between 1.5 and 2.o, (see ref. 2 I ) . At this state of the preparation the purity index was usually near 4.0. Further purification was obtained by chromatography on an ECTEOLA colunm, with the column volume at least four times the volume of the concentrated protein solution. The ECTEOLA (Mannex) was washed with I M NaOH in a large Buchner funnel until the washings were colorless. It was then washed with distilled water until the washings were neutral, followed by repeated washing with o.oi M phosphate buffer, pH 6. 9. The concentrated phycocyanin solution was placed on the column and eluted with phosphate buffer. Fractions with a purity index between 4.8 and 5.0 were collected, precipitated with saturated (NH4)2SO4, and stored at 6 ° in this form until used. Before use the precipitated protein was centrifuged, dissolved in the minimum w)t. of o.oi M phosphate buffer, pH 6.9, and dialyzed against three changes of dialysate at 6 °. The solution was then dialyzed against three changes of the corresponding 2H20 buffer (p2H 7-3) to ensure that all exchangeable protons were replaced by deuterium. The p~H of a buffer was calculated by adding o.4 to the reading obtained with a glass electrode pH meter calibrated with standard buffer in H20 22. The phycocyanin used in this study is an associating system of monomer, trimer, and hexamer 19,2a, and exists largely as trimer under the conditions of the experiments reported here 2a. Molar concentrations of protein used in this paper are referred to an assumed monomer molecular wt. of 4o ooo, based on amino acid analysis 2aand physicochemical measurements 2a. The sodium dodecyl sulfate was an Eastinan white label product, used without further tmrification. Buffer salts were reagent grade. The 99.8% 2H20 was obtained from the Savannah River Works of the Atomic Energy Commission and redistilled before use. Methods

Proton magnetic resonance spectra were obtained with a Varian HA-Ioo spectrometer. A capillary of benzene was used as the source of a lock signal and as an external reference. The chemical shift was converted to external hexamethyldisiloxane by subtracting the chetnical shift upfield from benzene (taken as positive) from 6.8o5 ppm. A Varian C-Io24 time averaging computer was used to improve signal-to-noise ratio for weak signals. Magnetic susceptibility measurements ~ indicated that susceptibility corrections for all the aqueous solutions used were identical within experimental error, so no corrections were made. When solutions in different solvents were compared, the usual corrections for bulk susceptibility were made 26. Visible and ultraviolet spectra were recorded on the Cary 14 spectrophotometer. Protein concentration was determined from the absorbance at the peak near 62o mff. Phycocyanin does not obey Beer's Law 20, and the absorption peak shifts with changes in concentration due to dissociation of the protein 2°, but the absorbance at the absorption maximum is a linear function of the protein concentration over the absorbance range o. 3 2.o. The extinction coefficient was determined by evaporating i%, weighed samples of protein to constant wt. in vacuum at 7o °. E ~ .... was found to be 68, in agreement with previous work 2°.

ldiochim. BioDh3,s. dcta, 175 (i969) 31-4 o

34

R. M. ROSENBER(;, H. L. CRESPI, J. J. KATZ

RESULTS

The proton magnetic resonance spectrum of sodium dodecyl sulfate has three well-defined main peaks and a fourth minor peak (Fig. I, a). The peak furthest downfield (4.362 ppm) is assigned to the C-I methylene, the peak at 1.624 ppm is assigned to the intermediate methylene groups, and the peak at 1.2oB ppm is assigned to the terminal methyl group. Area measurements indicate that the large peak at 1.624 ppm represents nine methylene groups, and the low, broad peak just downfield from this large methylene line represents the remaining methylene group. This assignment parallels that for n-decanol, the most closely related compound whose spectrum was found in the literature 27. On the addition of phycocyanin to o.oi M sodium dodecyl sulfate, the resonance peaks are shifted upfield and become broader (Fig. I, b, c, d). That all the main peaks 17

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Fig. I. P r o t o n m a g n e t i c resonance spectra of o . o i M s o d i u m dodecyl sulfate solutions in o . o i M p h o s p h a t e , p2H 7-3, w i t h v a r y i n g a m o u n t s of d e u t e r a t e d p h y c o c y a n i n . Probe t e m p e r a t u r e , 32°. E x t e r n a l b e n z e n e was used as the source of a lock signal and the scale is referred to external h e x a m e t h y l d i s i l o x a n e . The t o t a l areas are n o t c o m p a r a b l e in the four spectra, a, no p h y c o c y a n i n , 74 scans; b, 1 . 5 5 . i o - 4 M p h y c o c y a n i n , 225 scans; c, 4 . 6 5 . I o - 4 M p h y c o c y a n i n , 234 scans; d, 6.2o. l o -4 M p h y e o c y a n i n , 224 scans. Fig. 2. The c h e m i c a l shift of the p r o t o n m a g n e t i c resonance peaks of s o d i u m dodecyl sulfate as a f u n c t i o n of the ratio of m o l a r c o n c e n t r a t i o n of p r o t e i n to the m o l a r c o n c e n t r a t i o n of surfactant. Left scale: [Z, --(CH2)9 ; O, - - C H 3 . R i g h t scale: /~, C-I m e t h y l e n e . L o c k signal and reference as in Fig. i.

are shifted upfield provides evidence that the entire hydrocarbon chain of the surfactant interacts with the protein ; that the peaks are shifted upfield indicates that the surfactant is in a more hydrophobic environment when bound to the protein than when free in aqueous solution28,~9; and that the peak assigned to the C-I methylene beconles broadened at lower protein concentration than the other sharp peaks indicates that the adjacent ionic group of the surfactant is also involved in the interaction. Fig. 2 shows the chemical shift of the three prominent resonance peaks of sodium dodecyl sulfate as a function of the ratio of the molar concentration of protein to the molar concentration of surfactant. (The greater scatter for the C-I methylene Biochim. Biophys. Acla, 175 (1969) 31-4 °

SODIUM DODECYL S U L F A T E - P H Y C O C Y A N I N INTERACTION

35

resonance is due to the difficulty in choosing the peak position when the peak is broadened.) We interpret the plateaus in the curves to indicate that at surfactant to protein mole ratios between 12 and approx. 4 o, the surfactant is essentially completely bound, and that the plateau chemical shift value is characteristic of the bound state (Table I). The number 40 is in reasonable agreement with 36, the number of lysine and arginine residues per phycocyanin monomer unit 24. It would seem, then, that the surfactant is bound very strongly to the protein until all the positive sites on the protein are filled, and is less strongly bound at higher surfactant concentrations, where only the hydrophobic interaction is possible.

TABLE

I

CHANGES IN CHEMICAL SHIFTS OF SODIUM DODECYL SULFATE PEAKS DUE TO BINDING BY PHYCOCYANIN

Peak

Chemical shift (ppm)*

--CH~-(CH2) 9 ---CH a

Free

Bound

4.362 -4- o . o i 1.624 ~ o . o i 1.2o8 ± o . o i

4.255 1.525 i o . o t 1.145 d= 0.02

O.lO 7 0.099 --0.063

* L o c k s i g n a l a n d r e f e r e n c e a s in Fig. i. N o c o r r e c t i o n s f o r b u l k s u s c e p t i b i l i t y .

The largest resonance peak in Fig. i, d, in which the sodium dodecyl sulfate is essentially completely bound, is surprisingly narrow (approx. 0.28 ppm) for a molecule bound as firmly as we have postulated to a protein of molecular wt. 12o ooo, or even to protein monomer of molecular wt. 4 ° ooo. The narrow peak width suggests that the protein is denatured in these solutions, and that the peptide chains to which the detergent is bound must have much greater freedom of motion than they do in the native protein. Proton magnetic resonance spectra of phycocyanin in which 1H-leucine has been incorporated by biosynthesis indicate leucine methyl peak widths of approximately 0.50 ppm 3°. Fig. 3 shows the ultraviolet-visible absorption spectrum of phycocyanin (6.00lO -6 M) in buffer, with added I - l O -3 M sodium dodecyl sulfate, and with added I • IO-2 M sodium dodecyl sulfate. The striking changes in the absorption spectrum are comparable to those produced by other classical denaturing agents such as 8 M urea 31 or acidic ethanol 32, or by thermal denaturation20m. Fig. 4 shows the absorbance of the three main peaks in the ultraviolet-visible absorption spectrum of 6.20. IO 6 M phycocyanin as a function of sodium dodecyl sulfate concentration; the values on the vertical coordinate axis are those of the protein in buffer alone. To test whether the spectral changes observed are a result of change in protein conformation, and not merely a result of a changing the microenvironment of the chromophore through addition of surfactant, the spectrum of the free ehromophore phycocyanobilin 34 was studied as a function of sodium dodecyl sulfate concentration. Fig. 5 shows the absorption spectrum of phycocyanobilin in buffer and in 4.8. Io -2 M sodium dodecyl sulfate. It can be seen that the effect of the Biochim. Biophys. Acta, 175 (1969) 3 1 - 4 °

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F i g . 3. A b s o r p t i o n s p e c t r a o f p h y c o c y a n i n , 0 . o o . [o 6 M, i n o . o l M p h o s p h a t e , p°-H 7.3. b u f f e r ; . . . . , i . o " IO -a ~ i s o d i u m d o d e c y l s u l f a t e ; l . o - [ o -2 M s o d i u m d o d e c y l A f t e r 2~ h a t 25 ° .

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F i g . 4. A b s o r b a n c e o f t h r e e m a i n p h y c o c y a n i n p e a k s as f u n c t i o n o f s o d i u m d o d e e y l s u l f a t e ( S I ) S ) c o n c e n t r a t i o n . 6 . 2 0 • IO 6 ~'I p h y c o c y a n i n in o . o i M p h o s p h a t e , p2H 7.3. ( ) , ,, 6 2 0 m u, ~ • . - [-~, 36omff, ~ - L \ , 2 8 o m f f A f t e r 2 4 h a t 25 °.

surfactant on the three peaks is opposite to the effect on the spectrum of phycocyanin (Fig. 3). The absorbance of the three main peaks of the phycocyanobilin spectrum as a function of sodium dodecyl sulfate concentration is shown in Fig. 6, where it can be seen that changes in the free chromophore spectrum occur at a much higher surfactant concentration than in the protein, in addition to being in the opposite direction. Thus, we take the spectral data to indicate a change in the conformation of the protein. If our conclusions from the line-width of the --C H 2 - proton magnetic resonance 14 1.3 1.2

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Biochim. Biophys. dcta,

T75 ( I 9 0 9 ) 31 4 o

SODIUM DODECYL SULFATE-PHYCOCYANIN

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INTERACTION

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F'ig. 6. T h e a b s o r b a n c e a t t h e a b s o r p t i o n p e a k s o f p h y c o c y a n o b i l i n a s a f u n c t i o n o f s o d i u m d o d e c y l s u l f a t e (SDS) c o n c e n t r a t i o n in o . o i M p h o s p h a t e , p 2 H 7.3, a f t e r 24 h a t 25 °. 2.55 • IO -3 g / i o o m l ; e q u i v a l e n t p r o t e i n c o n c e n t r a t i o n , 1 . 6 . i o - s M. @ 0 , 5 9 8 m # p e a k ; [ ] . . . . E~, 361 m/~ peak; ~ - ---~, 275 m/~ p e a k .

p e a k are correct, b o u n d s o d i u m d o d e c y l sulfate below a c o n c e n t r a t i o n of s u r f a c t a n t of a b o u t 4.2. IO -4 M should show a b r o a d e r resonance p e a k t h a n b o u n d s u r f a c t a n t at higher c o n c e n t r a t i o n s at which t h e p r o t e i n is d e n a t u r e d (Fig. 7). A l t h o u g h the large m e t h y l e n e p e a k is c l e a r l y seen in 2 • I o -~ M s o d i u m d o d e c y l sulfate in t h e absence of protein, the a d d i t i o n of 1.55- I o 4 M p h y c o c y a n i n is sufficient to b r o a d e n the s u r f a c t a n t resonance p e a k to t h e e x t e n t t h a t it is no longer observable. This p r o t e i n c o n c e n t r a t i o n is t h e same as used in the e x p e r i m e n t shown in Fig. I, b, where a s h a r p resonance p e a k was seen. T h a t t h e resonance p e a k of s o d i u m d o d e c y l sulfate b o u n d to n a t i v e p r o t e i n

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F i g . 7. P r o t o n m a g n e t i c r e s o n a n c e s p e c t r a o f s o d i u m d o d c c y l s u l f a t e in 2 - 3 " l o 4 M s o l u t i o n s , o . o t M p h o s p h a t e , p 2 H 7.3. P r o b e t e m p e r a t u r e , 320 . a, 2 . o - l o -~ M s o d i u m d o d e c y l s u l f a t e , 0 7 3 s c a n s ; b, 2.85 • I o -4 M s o d i u m d o d e c y l s u l f a t e in ~.55 " i o 4 M p h y c o c y a n i n , 931 s c a n s . L o c k s i g n a l a n d r e f e r e n c e a s i n F i g . 1. Fig. 8. P r o t o n m a g n e t i c r e s o n a n c e s p e c t r a o f I . O . I O -';I ~'I s o d i u m d o d e c y l s u l f a t e in o . o i M p h o s p h a t e , p 2 H 7.3- P r o b e t e m p e r a t u r e 32°. a, n o p r o t e i n , 9 8 o s c a n s ; b, 7.75 • lO-S M p h y c o c y a n i n , ~ooo s c a n s ; c, 1.55 • IO -~ M p h y e o c y a n i n , i o o o s c a n s . L o c k s i g n a l a n d r e f e r e n c e a s i n F i g . i. Biochim. Biophys. dcla, i75 (i969) 31-4 °

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is even broader than the resonance peak of biosynthetically incorporated leucine 3' lends support to the conclusion drawn from optical rotation n, spectrophotometric 16, and optical rotatory dispersion data'~J 4 that the protein structure is tightened by the binding of small amounts of surfactant. The proton nlagnetic resonance spectrum of an intermediate concentration of sodium dodecyl sulfate (I.O.IO a M) is shown in Fig. 8. At this surfactant concentration, the proton resonance peaks are observable at a surfactant to protein mole ratio as low as 7, indicating a transition between the tightening effect at very low surfactant concentrations and the denaturing effect at high surfactant concentrations. This conclusion is confirmed by the absorption spectrum of phycocyanin in these solutions. The absorption spectrum is changed very little at I • IO 3 M surfactant, yet the proton magnetic resonance spectrum is clearly seen. The proton magnetic resonance spectrum of lO -4 M sodium dodecyl sulfate cannot be observed even in dilute protein solutions because the peaks are broadened by the tight binding of the surfactant to the protein at low surfactant to protein mole ratios (Fig. 7). It was possible, however, to observe a very broad resonance peak of bound sodium dodecyl sulfate at very high protein concentrations (above 1.6. IO-3 M), where even o.o2 M surfactant does not denature the protein (as indicated by the persistence of the characteristic red fluorescence 19, since the solution was too highly light absorbing to record absorption spectra). The maximum of the broad resonance peak is observed to occur at 1.2 ppm, and the results for five such solutions are shown '

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F i g . 9- Chemical shift of the largest resonance peak of s o d i u m dodecyl sulfate (SDS) as a f u n c t i o n o f protein to s u r f a c t a n t mole ratio. L o c k signal and reference as in F i g . I. ~ , protein c o n c e n t r a tions less t h a n 8 • IO 4 M ; m, p r o t e i n c o n c e n t r a t i o n s greater t h a n 1.6 • IO -3 M. F i g . IO. T e m p e r a t u r e d e p e n d e n c e of c h e m i c a l shift, a - - A , m e t h y l e n e p e a k of free s o d i u m dodecyl sulfate in buffer; A - - A , m e t h y l e n e p e a k of p r o t e i n - b o u n d s o d i u m dodecyl sulfate in buffer; 0 , m e t h y l e n e p e a k of n - b u t a n o l in buffer; C ) - - - @, m e t h y l e n e p e a k of n - b u t a n o l in C~2HI~; m - - m , O H p e a k o f n - b u t a n o l in buffer; [ 3 - - [ ~ , O H p e a k o f n - b u t a n o l in C62H12. Chemical shifts in C62H12 include a b u l k susceptibility correction o f - - 0 . 0 3 3 p p m , corrected to the b e n z e n e reference. Chemical shifts in buffer include a b u l k susceptibility correction o f - - o , 2 2 6 p p m , corrected to the b e n z e n e reference. Biochim,

Biophys. Acta, 175 ( I 9 6 9 ) 31 4 °

SODIUM DODECY'L SULFATE-PHYCOCYANIN

INTERACTION

39

in Fig. 9, together with the data from the uppermost curve of Fig. 2. The surfactant to protein mole ratios for points on the lower line in Fig. 9 vary from 3.6 to 13.9. These results indicate that the tightly bound dodecyl sulfate experiences a much larger upfield shift than the surfactant more loosely bound to the denatured protein. Since the range of protein to surfactant mole ratios of the two curves in Fig. 9 overlap, it is also likely that the interaction with protein at higher protein concentrations is distinctly different from that at lower concentrations. Th~ difference m a y be that between interaction of the surfactant with the protein hexamer at high concentration as opposed to interaction with the protein trimer at low concentrations 2°, but no studies of the molecular weight at these high concentrations has so far been possible. We concluded from the results shown in Fig. I and from results in the literature on solvent effects28, 29 that the upfield change in the chemical shift of the resonance peaks of sodium dodecyl sulfate is due to a transfer to a more hydrophobic environment. To test this conclusion further, we measured the chemical shift of the largest resonance peak of free and bound sodium dodecyl sulfate as a function of temperature, together with the methylene resonance peak of n-butanc! in buffer and in C6~H12, and the OH resonance peak of n-butanol in buffer and in C62H1~. The results are shown in Fig. IO. The upfield change in chemical shift due to binding is seen to be essentially independent of temperature, with the chemical shift of free and bound surfactant increasing with increasing temperature. Although the chemical shift of the methylene resonance of n-butanol in buffer increases with increasing temperature~ as with dodecyl sulfate, the chemical shift in the hydrophobic solvent decreases with increasing temperature. This effect of the hydrophobic solvent is seen eveL more strikingly in the data for the OH group of normal butanol shown in the upper part of Fig. IO (note the change in scale). The chemical shift in buffer decreases with increasing temperature, and this temperature dependence is greatly accentuated on being dissolved in a hydrophobic solvent. Our conclusion that bound dodecyl sulfate is in a more hydrophobic environment when bound to the protein than when free in aqueous solution must be modified, then, to indicate that it is still in an "aqueous" hydrophobic environment, and not in an environment entirely comparable to an organic solvent. DISCUSSION

The data presented here on tile chemical shift of the proton magnetic resonance peaks of sodium dodecyl sulfate bound to phycocyanin provide the first direct evidence for the interaction of the entire hydrocarbon chain of the surfactant with protein. We thus confirm the indirect evidence on this point provided by studies of the effect of chain length on binding affinity 3-5 and conformational changeS,14,1~. Similarly, the extremely broadened resonance peak of the surfactant bound at low mole ratios of dodecyl sulfate to protein provides direct evidence for a tightening of protein structure when dodecyl sulfate is bound to a few high affinity sites. Such tightening of structure has previously been inferred from changes in optical rotationS,l°,11,14, titration behavior 16, and protection against urea denaturation13, ~5. From the upfield change in chemical shift of the dodecyl sulfate resonance peaks on being bound to protein, we concluded that the surfactant is in a more hydrophobic environment when bound to the protein than when free in aqueous solution. This conclusion must be qualified, however, by the observation that the chemical shift of Biochim. Biophys. Acta, i 7 5 (1969) 31 4 °

~[()

R. ,M. I¢(ISI2NBIcI¢{;, H. I.. ( ' R E s l q , J. J. l,Z\lZ

bounct dodecvl sulfate increases with increasing temperature, of the corresponding temperature.

resonance

peak of n-butanol

The environment

to be sure, more hydrophobic hydrocarbon.

of the bound

while the chemical shift

in Cg'~Hle d e c r e a s e s w i t h i n c r e a s i n g

surfactant

provided

by the protein

Silnilar conclusions were reached about the environment

micelle from measurements soapaG, aT.

is,

t h a n a n a q u e o u s s o l u t i o n , b u t it is n o t t h a t ofm~. a l i p h a t i c in a d e t e r g e n t

o f t h e 1917 c h e m i c a l s h i f t o f a C F a s u b s t i t u t e d

carboxylate

A('KNOWLED(;EMENT This Energy

work

was performed

Commission.

Division of Nuclear appreciate NMR

Education

the expert

spectrometer

spectrophotometric

under

the auspices

One of us (R.M.R)acknowledges technical

and Training assistance

and the assistance

of the support

of the Atomic

United

States

Atomic

from Grant 1622 of the Commission.

We

o f M i s s G A l L NORMAN in o p e r a t i n g

Energy

the

o f M i s s MARJORIE BURGESS w i t h p r e l i m i n a r y

studies. We acknowledge

h e l p f u l d i s c u s s i o n s w i t h R . LUMRY.

REFERENCES M. L. ANSON, .]. (;en. Physiol., 23 (1939) 239. F. W. PUTNA.XI AND H. NEURATH, J. Biol. Chem., J59 (t945) 195. F. I'2ARUSH AND M. SONENBERG, J. Am. Chem. Soc., 7~ (19491 1309. A. RAY, J. A. REYNOLDS, H. POLET AND J. STEINHARDT, Biochemist13', 5 (19601 2{~o(). J. A. REYNOLDS, S. HERBERT, H. I'OLET AND J. STEINHARDT, Biochemistry, 6 (r967) 937. J. T. YaNG AND J. 12. FOSTER, J. Am. Chem. Soc., 75 (19531 5560. M. J. I'.aH.aNSCH AND I). R. BRIGGS, J. Am. Chem. Soc., 76 (1954) 1396. R. M. HILL AND ]). 1~. BRIOGS, J. Am. Che~lz. Soc., 78 (t956) 159o. J- F. FOSTER AND K. AOKI, J. A m . Chem. Soc., 80 (1958) 52i 5. J. SEDLAK, tJ. A. Thesis, W e s l e y a n U n i v e r s i t y , i950. G. MARKUS AND F. KARUSH, J. Am. Chem. Soe., 79 (1957) 3204 . B. JIRGENSONS, Arch. Biochem. Biophys., 94 (196t) 59. M. L. MEYEt~ AND \V. KAUZMANN, Arch. Biochem. Biophvs., 99 (L9021 3.I8 B. JIRGENSONS, J. Biol. Chem., 242 (I9671 912. H. POLET AND J. STEINHARDT, Biochemistry, 7 (t908) t348. R. 1,OVR~EN. J. Am. Chem. Soc., 85 (I9631 3677 . R. M. ROSF.NBERG AND I. M. KLOTZ, .[. A*t'I. Chem. Not., 77 (19551 259°. H. F. DABO1.L, H. L. CRESPI aND J. J. I'(ATZ, Biotech. Bioe~lg., 4 (~962) 28I. 1). S. BERNS, H. L. CI{ESpI .aND J. J. l,~aTZ, J. Am. Chem. Sot., 85 (19(13) 8. A. HaTTORI, H. L. CRESPI aND J. J. KaTz, Biochemistry, 4 (I965) 1213. T. T. BANNISTER ,Arch. Biochem. Biophys., 49 (~954) 222. A. K. COVINGTON, M. PAABO, R. A. ROBINSON .aND R. (;. BATES, Anal. Chem., 4 ° (i968) 700. A. HATTORI, H. L. CRESPI AND J. J. NATZ, Biochemistry, 4 (1965) 1225. B. T. COPE, U. SMITH, H. L. CRESPI aND J. J. KATZ, Biochim. Biophys. Acta, 133 (I967) 446 . K. ~:i'REI AND H. J. BERNSTEIN, J . Chem. Phys., 37 (1902) I89t. J. A. POPLE, \V. G. SCHNEIDER AND H. J. BF.RNSTEIN, High Resolutio~z Nuclear 31ag~zetic Resonance, McGraw-Hill Book Co., New York, 1959, p. 81. 27 NIWR Spectra Cataloy, V a r i a n Associates, 1962. -~8 M. J. STEPHEN', Mol. Phys., I (I958) 22:3. 29 C. I,. BELL AND S. S. DANYLUK, ,l. Ani. Chem. Sot., 88 (1966) 2344. 3 ° H . I,. CRESPI, R. M. ROSENI~ERG, AND J. J. KATZ, Science, 16l (t968) 795. 31 L. J. BOUCHER, H. L. CRESPI AND J. J. N.aTZ, Biochemistry, 5 (19601 3796. 32 C. OHEOCHA, Biochemistry, 2 (1963) 375. 33 J. J. KaTz, H. L. CRESPI .aND A. J. FINKEL, Pure Appl. Chem., 8 (t964) 471. 34 H. L. CRESPI, U. SMITH AND J. J. KATZ, Biochemistr% 7 (t968) 2232. 35 G. MARICUS, R. L. LOVE AND F. C. X,VlSSLER, .]. Biol. Chem., 239 (19641 3687 . 36 N. MULLER AND R. H. BIRKHAHN, J. Phys. Chem., 7 t (1967) 957. 37 N. MULLER AND R. H. BIRKHAHN, J. Phys. Chem., 72 (19681 583 . J 2 3 4 5 6 7 8 9 lo ll 12 i3 14 15 l(i 17 ~8 19 2o 21 22 23 24 25 26

Biochim. Biophys. Acta, 175 (1960) 31 4 °