Spectroscopic characterization of β-lactoglobulin-retinol complex

28

Biochimica et Biophysica Acta, 6 2 5 ( 1 9 8 0 ) 2 8 - - 4 2 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 38509

SPECTROSCOPIC CHARACTERIZATION OF ~-LACTOGLOBULIN-RETINOL COMPLEX

R O B E R T D. F U G A T E * a n d P I L L - S O O N S O N G **

Department of Chemistry, Texas Tech University, Lubbock, TX 79409 (U.S.A.) (Received J a n u a r y 31st, 1980)

Key words: ~-Lactoglobulin-retinol complex; Fluorescence; Rotational relaxation time; Circular dichroism

Summary 1. The absorption spectrum of retinol when bound to fi-lactoglobulin is vibrationally resolved. The circular dichroism spectrum exhibits the same structure, as does the fluorescence excitation spectrum. 2. Two molecules of retinol are bound per protein dimer, with a binding constant (Kd) of 2 • 10 -s M. Also, by fluorescence titration it was found that the m o n o m e r binds one molecule of retinol with essentially the same K d. 3. Energy transfer occurs from t r y p t o p h a n (donor) to retinol (acceptor) with a rate constant, k, of 4.4 • 10 s s -~. The distance between the centers of mass of the transition is 34 ~, corresponding to the energy transfer efficiency of 44%. 4 . The fluoresence lifetime of retinol increases dramatically on binding to /~-lactoglobulin, from approx. 2 to approx. 10 ns, as does the fluorescence quantum yield. 5. The retinol binding to fi-lactoglobulin does not show a pH dependence and the binding site is hydrophobic. 6. On the Sephadex G-100 column, retinol is chemically modified to a retro derivative which binds even more strongly to fi-lactogiobulin than does retinol. 7. The fi-lactoglobulin-retinol complex rotates anisotropically in solution with a fast (3 ns) and a slower (12 ns) component. This may be attributed to retinol being bound at a flexible region of the protein, where only segmental flexibility is observed, weighted by its proximity to one of the major axis rotational times.

* Present address: SLM Instruments, U~bana, IL 61801, U.S.A. ** To whom correspondence regarding this paper should be addressed.

29 Introduction

~-Lactoglobulin forms a complex with all-trans-retinol [ 1]. Interestingly, the complex shows some vibrational resolution in the absorption spectrum of the latter which does not appear even under low temperature (e.g., 77 K) conditions for the c h r o m o p h o r e itself [2,3 ]. The utility of retinol as a membrane probe has already been demonstrated [4] and as a fatty acid analog, as have other polyenes, most notably parinaric acid [5,6]. Binding o f retinol to/3-1actoglobulin induces not only structure in the absorption spectrum, b u t increases in the fluorescence quantum yield (¢F) and lifetime (TF). Our goal was to characterize the m o d e of binding and determine the binding parameters for the complex. During the course of this work, it became apparent that under certain conditions, retinol was chemically modified to a comp o u n d that displaced retinol in the protein. This complex was examined and the chemical identity was investigated b y various spectroscopic techniques. Materials and Methods

Mate rials All chemicals were obtained from Sigma Chemical Co. unless otherwise noted. Retinal was purified b y silica gel thin-layer chromatography. [3H]Retinol was prepared b y the reduction of retinal with KBT4 (Ventron) in ethanol. Retinol-binding protein was a gift from Professor J. Churchich. fi-Lactoglobulin solutions were made in 0.05 M potassium phosphate buffer at pH 5.8 according to Piez [7], after DEAE-cellulose chromatographic fractionation of the protein. The A and B genetic variants of fi-lactoglobulin were eluted with a linear gradient of NaC1 from 0 to 0.08 M in 0.05 M phosphate buffer, pH 5.8. Methods fl-Lactoglobulin modifications: Removal o f histidine 159: The modification of the protein by removing the C-terminal isoleucine and the penultimate histidine with carboxypeptidase A has been reported to occur much more rapidly than hydrolysis at other sites [ 8,9]. fl-Lactoglobulin was dissolved in 0.2 M NaC1 unbuffered at pH 8.0 and 2500 U carboxypeptidase A (Sigma) were added and allowed to react for 3 h at 37°C. The reaction mixture was centrifuged at 3000 × g for 10 rain. The supernatant was then adjusted to pH 5.6 with 1 M HC1, and the crystalline suspension which immediately formed was dialyzed overnight at 4°C. The purified crystalline material was finally dissolved in 0.1 M phosphate, pH 7.5. The number of histidines remaining was evaluated b y selective carbethoxylation using diethylpyrocarbonate [10]. The number of histidines reacted was calculated using a Ae of 3200 M -~ cm -1 [10--12] per imidazole and measuring the absorbance decrease at 242 nm. Tryptophan modification. 2-Hydroxy-5-nitrobenzyl bromide (HNBB) reacts specifically with t r y p t o p h a n under neutral or acidic conditions [3]. This reaction was carried o u t at pH 2.0 in 0.4 M NaC1. The reaction was

30 immediate and the half-life of HNBB in water was approximately 1 min [14]. To quantitate the number of modified residues, the dialysate was diluted 15 times and made to a pH greater than 10 by adding 1 M NaOH. The solution turns bright yellow. An extinction coefficient of 1.8 • 104 M -1 • cm -1 at 410 nm has been reported [15] and was utilized to calculate the number of tryptophans modified. The absorption spectrum of the HNBB derivative showed ~max at 410 nm. Preparation o f anhydrovitamin A. Anhydrovitamin A was prepared by the literature m e t h o d [16,17]. Its absorption spectrum was measured and it agreed with the literature spectrum [17,18]. The yield of trans-anhydrovitamin A was less than 10%. Retroretinyl acetate preparation. Retroretinyl acetate was synthesized by the m e t h o d of Beutel et al. [19]. The absorption spectrum of the retroretinyl acetate prepared agreed with the literature absorption spectrum [ 19 ]. Stoichiometry determination. Stoichiometry was determined by fluoresence titration and radioisotope labelling. Both methods are described in the section that follows. Retinol was labelled by reducing retinal with KBT4, yielding all-trans-3H (15-C)retinol which was subsequently purified by TLC. The final material had a specific activity of 3.4 Ci/mol. This was added to fi-lactoglobulin solution, and then separated on Sephadex G-100, and 0.1 ml of each fraction was counted in 10 ml of liquid scintillation cocktail [20]. Corresponding protein concentrations were determined by the m e t h o d of Bradford [21] as commercially available from BioRad (Richmond, CA). The fluorescence titration was done on an Aminco-Bowman spectrofluorimeter at room temperature. Retinol in ethanol was added to 2.0 ml of ~-lactoglobulin containing 228 nmol protein in 0.1 M phosphate, pH 7.5, and the intensity was recorded. The fluorescence of the free retinol a m o u n t e d to only 2--3% of the complex fluorescence, but was nonetheless corrected for in the analysis. The analysis was essentially that of Cogan et al. [22] outlined herein. From mass law considerations, g d =

n [fl-lactoglobulin] [retinol] [fi-lactoglobulin • retinol]

(1)

where n is the number of sites on fl-lactoglobulin. Now if F denotes the fraction of free binding sites and [fl-lactoglobulin]o and [retinol]o the total protein and retinol concentrations, respectively, Kd can be expressed as follows: F Ka - 1 -- F

[retinol] o -- n [~-lactoglobulin] o (1 -- F)

(2)

which can be transformed to linearity to give [fl-lactoglobulin] o F -

1 [retinol]o F n (1 -- F)

Kd n

(3)

Plotting this equation gives a straight line with a slope equal to 1In and an intercept o f - - K a / n . Spectral and nanosecond fluorescence measurements. Absorption spectra

31 were measured on a Cary 118C spectrophotometer. CD and fluorescence spectra were recorded on a JASCO CD-ORD spectropolarimeter and a Perkin-Elmer MPF-3 spectrofluorimeter, respectively. The quantum yield o f fluorescence for the complex was measured relative to that o f all-trans-retinol (¢F = 0.008), after correcting for refractive index differences of ethanol and aqueous buffer as measured on an Abbe refractometer. Fluorescence polarization (P) or anisotropy (r) was measured on an SLM 480 phase-modulation fluorimeter in the polarization mode. Polarization bias due to instrumental artifacts was effectively eliminated b y the 'double-ended', simultaneous detection of parallel and perpendicular components of fluorescence with t w o photomultiplier tubes [24]. Fluorescence lifetime (rF)Was also measured o n t h e SLM 480 phase modulation fluorimeter. Nanosecond fluorescence measurements were also carried o u t on an instrument constructed according to Ref. 25. Fluorescence decay data were treated on a c o m p u t e r such that the effects of the lamp flash on the sample decay curve were effectively subtracted out. The m e t h o d utilized in this system was the phase-plane m e t h o d [26]. The boxcar averager system used can be used for recording nanosecond time-resolved fluoresence spectra [27]. Rotational relaxation time. The SLM phase-modulation fluorimeter can also be used for measurement of the time
r(t)=roe

--Pt

3

(4)

where p is rotational relaxation time. In the phase lag m o d e of measurement, p can be obtained from solving the quadratic equation [30] 2c~' (1 - - I Po a': + 3 --P-----~o ta~

I

)

COrE +

( 1 + co:v~)(1 - - P : o ) = O (3 - - P o ) :

(5)

where ~'= TF/p and P0 is the limiting degree of polarization. The measured anisotropic phase lag, tan A, is given by tan 5 t t - tan 51 tan A = 1 + tan ~iHtan 8±

(6)

---- LO A T F

where 5 Ir and ~i± are phase lags of the parallel and perpendicular components, respectively. The boxcar averager unit can also be used to evaluate rotational relaxation time b y pulse fluorimetry [31]. Results

Fig. 1 shows the absorption spectra o f all-trans-retinol in ethanol and the fi-lactoglobulin-retinol complex. The absorption spectrum of the complex shows vibrational structure which is absent in retinol even under low temperature (77 K) conditions. The extinction coefficient relative to that in ethanol (ea24nm = 45 710) is alSO decreased (eaasnm = 34 830). Fig. 2(A) shows the CD spectrum of the fi-lactoglobulin-retinol complex, showing the vibrational structure corresponding to that in the absorption Spec-

32 ~ 0.4

A

/P

0.3

/

0.2 0.1 0

A

/' /

' 250

250

300

350

300

350

400

400

B

Jo o.3

B

(_D

0.2 0.1

3OO

'

4--00

i

5o0

-I0

250 550 450 WAVELENGTH, nm

-15 250

350

450

Wovelengfh, n m

Fig,. 1. A. T h e a b s o r p t i o n s p e c t r u m o f all-trans-retinol in e t h a n o l ( b l u e - s h i f t e d s p e c t r u m ) a n d j3-1actoglob u l i n - r e t i n o l c o m p l e x in 0.1 M p o t a s s i u m p h o s p h a t e b u f f e r , p H 7 . 5 , 2 9 8 K . C o n c e n t r a t i o n o f r e t i n o l in e t h a n o l is 9 . 7 4 tiM, a n d in t h e t3-1actoglobulin c o m p l e x is 1 2 . 9 tzM. P r o t e i n c o n c e n t r a t i o n is 1 1 4 tiM. F o r r e t i n o l , e t h a n o l w a s t h e r e f e r e n c e a n d f o r t h e c o m p l e x , t h e s a m e c o n c e n t r a t i o n o f p r o t e i n . B. T h e a b s o r p t i o n s p e c t r u m o f t h e ~-lactoglobulin-all-trans.retinol ( ' D ' ) r e s o l v e d d i m e r in 0.1 M p h o s p h a t e b u f f e r , p H 7.5 2 9 8 K; r e t i n o l c o n c e n t r a t i o n is 1 . 0 6 • 1 0 -$ M. C. T h e n - h e x a n e e x t r a c t o f t h e r e s o l v e d ~ - l a c t o g l o b u l i n r e t i n o l 'D' c o m p l e x f r o m t h e G - 1 0 0 c o l u m n (el. lVig.'6).

Fig. 2. A. T h e circular d i c h r o i s m s p e c t r u m o f ~ - l a c t o g l o b u l i n ( ) a n d its r e t i n o l c o m p l e x , r u n at 1 1 4 tiM a n d 1 0 0 - t i m e s d i l u t e d f o r t h e far-ultraviolet r e g i o n ( . . . . . . ). B. T h e CD s p e c t r u m o f all-trans retinolr e t i n o l b i n d i n g p r o t e i n c o m p l e x . C. T h e C D s p e c t r u m o f t h e r e s o l v e d 'D' c o m p l e x o f ~3-1actoglobulin.

trum. Retinol-binding protein-retinol complex shows the opposite CD sign for the main absorption band (Fig. 2(B)). As the pH is lowered below 3.5, the 36 000 dalton dimer dissociates into two identical 1 8 0 0 0 dalton subunits (monomer) [32--35]. The fi-lactoglobulin A variant also forms a tetramer at pH between 3.7 and 5.1 and at less than 20°C

[35]. At pH 2 (i.e., monomer), the absorption spectrum of the complex is slightly more resolved than at 7.5 (dimer), and there is a fairly well defined peak at 400 nm in the CD spectrum that was not observable in the CD of the dimer complex. From the use of [3H]retinol, and by knowing the protein concentration, retinol concentration and the correction factor for retinol interference in the protein assay, the binding ratio can be calculated, yielding the ratio of 1.98 -+ 0.06 mol ,retinol bound per mol fl-lactoglobulin. A Scatchard plot based on the indueed CD signal (Fig. 2) at 330 nm as a function of [retinol] also yielded 2 mol retinal per mol dimeric fi-lactoglobulin. The binding isotherm at pH 7.5 (dimer) ' based on the plot of fluorescence intensity vs. [retinol] and the Scatchard plot based on the induced CD signal at

33

~

~

! ',

0 CO LIJ nO

A

250

350

450

550

300

4o0

500

600

,,

01~ -

i

,

i

o

I

2

3

I

I

I

I

4

5

6

7

[refinol]o. F

WAVELENGTH,

nm

(i-F) F i g . 3. A. T h e b i n d i n g o f r e t i n o l to t h e f l - l a c t o g i o b u l i n m o n o m e r . T h e i n t e r c e p t h a s p r e v i o u s l y b e e n s h o w n t o be - - K d / n ( = - - 0 , 0 2 ) , c o r r e s p o n d i n g to K d o f 2 • 10 -8 M. T h e s l o p e y i e l d s n = 1. P r o t e i n c o n c e n t r a t i o n w a s 2 . 2 8 p M ( m o n o m e r ) in 0.1 M N a C I / H C l , p H 2 . 0 . B. T h e b i n d i n g o f r e t i n o l to t h e fl-lactogiob u l i n d i m e r . T h e slope y i e l d s n = 2. K d = 2 - 10 -8 M. P r o t e i n c o n c e n t r a t i o n w a s 1 . 1 4 #M in 0.1 M p h o s p h a t e b u f f e r , p H 7.5. Fig. 4. A. C o r r e c t e d r o o m - t e m p e r a t u r e e x c i t a t i o n a n d e m i s s i o n s p e c t r a o f t r a n s - r e t i n o l ( . . . . . . ) and flmlactogiobulin c o m p l e x ( ). C o n c e n t r a t i o n o f r e t i n o l w a s 2.19 p M in e t h a n o l a n d ~ - l a c t o g l o b u l i n c o n c e n t r a t i o n w a s 5 . 0 4 p M in 0.1 M p h o s p h a t e b u f f e r , p H 7.5. S p e c t r a l b a n d p a s s w a s 2 n m f o r t h e r e s p e c tive spectra. Excitation and emission wavelengths are d e n o t e d b y t h e a r r o w s . T h e e m i s s i o n s p e c t r a are i d e n t i c a l , e x c e p t f o r t h e i r q u a n t u m y i e l d . O n l y o n e is s h o w n h e r e f o r c l a r i t y . I n t e n s i t i e s in t h e u l t r a v i o l e t r e g i o n o f t h e e x c i t a t i o n s p e c t r u m o f t h e c o m p l e x are l o w e r t h a n t h o s e o f t h e a b s o r p t i o n s p e c t r u m d u e to i n c o m p l e t e c o r r e c t i o n f o r t h e w e a k X e l a m p o u t p u t in t h i s r e g i o n . B. T h e c o r r e c t e d f l u o r e s c e n c e e x c i t a t i o n a n d e m i s s i o n s p e c t r a o f t h e ' r e s o l v e d ' c o m p l e x ( d a s h e d line). A b s o r b a n c e w a s l e s s t h a n 0 . 1 5 t h r o u g h o u t t h e r e g i o n s c a n n e d . B a n d p a s s w a s 4 n m f o r all s p e c t r a s h o w n .

330 nm (Fig. 2) as a function of [retinol] showed no evidence of cooperative interactions between the two subunit binding sites, as they closely resembled that of monomeric ~-lactoglobulin at pH 2. From Fig. 3, the binding constant, Kd, was calculated to be 2 • 10 -8 M for both monomer and dimer. Fig. 4 shows the excitation and emission spectra of fl-lactoglobulin and its retinol complex. A large spectral overlap exists between the protein fluoregcence (not shown) and the retinol excitation spectrum. The excitation spectrum is vibrationally resolved and is in good agreement with the absorption spectrum. The emission spectrum is broad and structureless, however. The emission spectra of retinol and the complex are identical except for the quantum yield (OF = 0.008 for retinol and 0.033 for complex). Table I lists the absorption (or excitation), emission and CD maxima for trans- and 13-cisretinol in ethanol and their complexes. 13-cis-Retinol bears a remarkable resemblance to all-trans-retinol, with the exception of the emission maximum. The fluoresence lifetimes of retinol and the c o m p l e x are shown in Table II and Fig. 5. A dramatic increase in the fluorescence lifetime of retinot ( T F .'-~- 2.8

34 TABLE I C O M P A R I S O N O F A B S O R P T I O N , CD, E M I S S I O N A N D E X C I T A T I O N M A X I M A O F R E T I N Y L COMP O U N D S (IN E T H A N O L ) A N D T H E I R ~ - L A C T O G L O B U L I N (Lg) C O M P L E X E S ( p H 7.5) C o m p o u n d or c o m p l e x

hma x (nm) Absorption

All-trans r e t i n o l Lg-all-trans r e t i n o l 13-c/s r e t i n o l

Lg-13-cis r e t i n o l Anhydrovitamin A Lg-Anydrovitamin A retro retinylacetate

Lg-retro r e t i n y l a c e t a t e Lg-all-trans r e t i n o l ' D ' c o m p l e x

CD

325 360s, 3 4 2 330, 317s 325,315s 365s, 3 4 5 3 8 7 s , 367 349s, 330s * 392s, 377 356s 367s, 3 4 6 330 * 373s, 352s 336, 3 2 3 371s, 352 335

3 6 2 s , 343 330 363s, ~ 3 4 0

Emission 470 470 ~520 522 ~562 ~530 ~538 ~532 507

* E x c i t a t i o n m a x i m a ; s, s h o u l d e r .

ns) when b o u n d to the protein (10 ns for complex) is observed, in agreement with previous reports [9,10]. It has been shown that the retinol binding is not pH dependent in the range 2 to 7.5. From the red shift in the absorption spectrum, it can be inferred that the binding site is hydrophobic. Removal of the penultimate histidine had no observable effect on the bindT A B L E II F L U O R E S C E N C E L I F E T I M E S O F f l - L A C T O G L O B U L I N A N D T H E C O M P L E X E S , M E A S U R E D BY THE PHASE LAG METHOD

Sample

Conditions

~-Lactoglobufin (Trp)

pH 2 p H 7.5

AU-trans-Retinol

Complex (Trp)

ethanol pH 2 p H 7.5 p H 7.5

13-c/s R e t i n o l Complex (Retinol) Complex (Trp)

ethanol p H 7.5 p H 7.5

2.0 +_ 0,47 9.0 + 1.2 0 . 9 3 +- 0 . 3 2

Anhydrovitamin A Complex

ethanol p H 7.5

2.51 + 0 . 4 1 9 1.56 -+ 0 . 0 8 6

Retinyl acetate Complex

ethanol p H 7.5

1.53 +- 0 . 9 1 5 8 , 3 3 -+ 0 . 6 0 3

Retro r e t i n y l a c e t a t e

ethanol p H 7.5

0.28 + 0.10 1 2 , 3 3 _+ 0 . 6 8 3

hexane p H 7.5

1.68 + 0 . 1 4 0 10,01 + 0.423

Complex (Retinol)

Complex n-Hexane extract of 'D' Complex * Trp fluorescence polarization.

~'F (ns) 1.40 +- 0.17 1 . 4 0 _+ 0 . 1 6 2.81 17.00 10.50 0.87

+_ 0 , 7 3 +- 1 . 4 0 _+ 0 , 7 9 -+ 0 , 4 4

Polarization

0 . 3 0 1 _+ 0 . 0 0 9 *

0 . 2 9 8 -+ 0 . 0 0 8

0 . 2 3 4 -+ 0 . 0 1 0

0 . 3 2 8 -+ 0 . 0 0 9

0.006 + 0.010 0 . 3 0 1 -+ 0 . 0 0 9

35 4.24

~ 306 N

°°,~o *

o

I. 87

0,69

. . . . 0.160

-- , 0 1.3 7 W(t),

,

,

,

0.474

ns-I

Fig. 5. T h e phase-plane p l o t o f t h e f l u o r e s c e n c e decay o f 13-1actoglobulin-retinol c o m p l e x . T h i s is the d e e o n v o l u t e d d a t a f o r t h e c o m p l e x , e x c i t i n g at 3 4 9 n m a n d m o n i t o r i n g emission at o v e r 4 5 0 n m at r o o m t e m p e r a t u r e . T h e l i f e t i m e is 1 0 . 0 7 +- 0.1 ns. As can be seen, t h e t r a c e is l i n e a r , w i t h a c o r r e l a t i o n c o e f f i c i e n t o f 0 . 9 9 6 4 f o r t h e best l i n e t h r o u g h t h e p o i n t s .

ing of retinol to fl-lactoglobulin, as judged by the polarization value (P = 0.234 +- 0.002) and lifetime (TF ~ 10 ns) at room temperature. There are four t r y p t o p h a n s per dimer and the HNBB modification altered all four residues (found, 3.82 residues). Retinol is apparently still bound, as evidenced by the fluorescence polarization (P = 0.275 -+ 0.019) *. However, the vibrational structure disappeared and the absorption m a x i m u m of the complex was blue-shifted to approx. 325 nm, essentially t h a t of free retinol. Thus, modification o f the t r y p t o p h a n destroys the unique feature of fl-lactoglobulin to induce the vibrational structure of retinol. That this structure is unique is seen from binding of retinol to bovine serum albumin, h u m a n serum albumin [2] and the various retinol binding proteins [36--41] all of which exhibit no vibrationally resolved structure. The fluorescence lifetime becomes rather heterogeneous however, (at 30 MHz, ~F(8) = 7.76 +- 1.07, rF(M) = 10.79 ± 0.61

ns). The HNBB modification o f protein was also run under the same conditions, except that retinol was complexed to see if retinol 'protected' t r y p t o p h a n against modification by blocking the HNBB's access to tryptophan. Retinol did n o t protect the protein from modification; by a procedure identical to that reported [ 15 ], 3.79 residues were calculated to be modified, yielding the same spectrum as t h a t obtained for the HNBB-modified protein-retinol complex. The binding of retinol to fl-lactoglobulin under denaturing conditions was also investigated. In 8 M urea, pH 7.24, retinol still binds (P = 0.279 + 0.021) * to the protein. The absorption spectrum showed the loss of vibrational structure and the absorption m a x i m u m was blue-shifted to approx. 325 nm. Denaturation with SDS also had the same effect. This was surprising as it has been postulated t h a t fl-lactoglobulin under these conditions existed as a random coil [42,43]. However, the above result is consistent with the data for retinol* Alternatively, mieeUar formation of freed retinol can give rise to the high polaxization values, although this m a y be unlikely at the concentration of retinol involved.

36 1.0

306 g

'

<~0.4

F',,

o

0.2 0

5

I0 Fraction

15 Number

20

Fig. 6. ~-Lactoglobulin-all-trans-retinol c o m p l e x r e s o l v e d o n a S e p h a d e x G - 1 0 0 c o l u m n , 1 X 90 c m , at r o o m t e m p e r a t u r e . ~ - L a c t o g l o b u l i n at a c o n c e n t r a t i o n o f 4 m g / m l in 0.1 M p h o s p h a t e b u f f e r , p H 7.5 w a s p l a c e d on t h e c o l u m n a f t e r a d d i n g r e t i n o l t o a n a p p r o x i m a t e l y 2 0 - f o l d e x c e s s . T o t a l e t h a n o l c o n c e n t r a t i o n ( r e t i n o l s o l v e n t ) w a s less t h a n 3%. M, D a n d T s t a n d f o r a p p a r e n t m o n o m e r , d i m e r a n d t e t r a m e r , r e s p e c t i v e l y . T h e d o t t e d line s h o w s f l - l a c t o g l o b u l i n o n l y , r u n u n d e r t h e s a m e c o n d i t i o n s . I n s e t : A s e m i l o g a r i t h m i c p l o t o f m o l e c u l a r w e i g h t v e r s u s f r a c t i o n n u m b e r f o r t h e G - 1 0 0 c o l u m n , 1 X 90 c m . M o l e c u l a r w e i g h t s t a n d a r d s are b o v i n e s e r u m a l b u m i n , f l - l a a t o g l o b u l i n a n d l y s o z y m e , d e n o t e d b y e. T h e c o r r e s p o n d i n g / 3 - 1 a c t o g l o b u l i n - r e t i n o l c o m p l e x e s are d e n o t e d b y o.

binding protein, which still binds retinol in 6 M urea [40]. In order to characterize further the fi-lactoglobulin-retinol complexes, we attempted resolution of the mixture of fl-lactoglobulin and retinol. We applied the complex with excess retinol (10--30-fold) to a 1 X 90 cm Sephadex G:100 column. Fig. 6 shows the elution profile. Surprisingly, three peaks, nbt one, were eluted, as is the case when only fl-lactogtobulin (which comes 0utl at fraction 1 0 - 1 1 ) is passed through the column. The three peaks labelled' T, D i s n d M correspond to molecular weights of approx: 133 000, 37 000 and 19 0 0 0 , The nanosecond time-resolved emission spectra of the complex at 297:K in phosphate buffer showed that the emission curves were identical at all times, indicating that no dielectric relaxation of polar groups surrounding the chromophore occurred during the time scale investigated. The rotational relaxation time of molecules in solution may be evaluated from a knowledge of their fluoresence anisotropy as a function of time after excitation. From Eqns. 5 and 6, the approximate probe lifetime required for observations of the rotational relaxation time for fl-lactoglobulin can be calculated as 9.5 ns. Thus, this complex falls within the category of a useful system to study its hydrodynamics (i.e., the lifetimes of complexes are sufficiently long: cf. Table II). Discussion

1. Spectroscopic characterization of [J-lactoglobulin-retinol complex The absorption spectra of the fl-lactoglobulin-retinol complex have been described recently [44]. In the CD spectrum of the complex (Fig. 2), the vibrational structure in the chromophore region (300--400 nm) corresponds well to that in the absorption (Fig. 1) and fluorescence excitation (Fig. 4) spectra. The appearance of structure in the main absorption band and the increase in ~ F and Tv of the complex are attributable to a rigidity and a geometric change afforded to retinol when it binds to the protein [2,3].

37

2. Characterization o f the binding site The dimeric fl-lactoglobulin binds two molecules of retinol and the m o n o m e r binds one retinol molecule. These observations suggest that retinol tightly binds at specific sites on each subunit. It is interesting to note t h a t the same K d was calculated for both pH (2.0 and 7.5) conditions, implying that the retinol binding site in the protein contains no amino acid groups that ionize in the pH region [38--40,45]. In addition to retinol-binding protein, other proteins exhibit similar binding characteristics. For example, a retinol-binding protein which also exhibits a structured absorption spectrum and binds retinol tightly with K d = 1.6 • 10 -8 M has been isolated and characterized from rat liver [37]. From the red shift of ~maxand the appearance of vibrational structure in the absorption band of fl-lactoglobulin-retinol, it can be inferred t h a t the binding site enforces rigidity of the retinol molecule, especially the conformation of the ~-ionone ring relative to the polyene chain. The binding of the nonpolar end (fl-ionone) of retinol is likely to involv$ a hydrophobic site on each subunit. The m o n o m e r and dimer have such hydrophobic sites which can be occupied by alkanes, each subunit binding one alkane molecule [46--48]. To induce~the vibronic resolution in the absorption spectrum of the complex (Fig. 1), the binding site m a y encompass the fl-ionone and one or more adjacent C= C bonds. It is possible t h a t the binding site includes t r y p t o p h a n residues. There are two t r y p t o p h a n residues per subunit in a hydrophobic environment, and t h a t at least one is somewhat affected by the state of subunit association [49]. The present work indicates t h a t the tryptophans occupy similar environments and for the dimer (pH 7.5) # m o n o m e r (pH 2) equilibrium, the environment of t r y p t o p h a n is not significantly affected, as inferred from similar r r values for tryptophans (Table II). This explains the lack of pH effect on the K d of the complex. It may be recalled that the HNBB modification of all four t r y p t o p h a n residues in the dimer led to the loss of vibrational structure of the complex. The absorption m a x i m u m shifted to 325 nm, essentially t h a t of free retinol. On the other hand, the emission m a x i m u m slightly red shifted to 510 nm. These data thus indicate t h a t t r y p t o p h a n residue(s) is involved in the retinol binding site, possibly in the role of fixing the fi-ionone ring specifically with respect to the polyene chain. The denaturation abolishes the vibrational structure, but retinol still binds to fi-lactoglobulin. Retinol-binding protein, too, binds to retinol in 6 M urea [40]. From the CD spectrum of the retinol complex (Fig. 2), the involvement of t r y p t o p h a n residue(s) in the binding site can also be inferred. An approximate value of the rotational strength (R) of the chromophore CD band (Fig. 2) induced by the binding at an optically assymmetric site of fl-lactoglobulin was calculated to be --3.22 • 10 -39 (c.g.s.). This large R value can be interpreted in terms of optical activity arising from a Kirkwood-type coupled oscillator mechanism [50]. This could be envisioned as coupling of the retinol oscillator with that o f t r y p t o p h a n at or near the binding site, thus accounting for the strong induced CD of the complex. The fluorescence excitation spectrum of the complex has a shoulder on the blue edge of the main excitation band (Fig. 4). Comparison. with the absorption spectra o f retinol, p-lactoglobulin and the retinol complex showed that this

38 shoulder was due to t r y p t o p h a n residues, and appeared in the excitation spectrum as the result of energy transfer from t r y p t o p h a n to the retinol. An excellent overlap between the t r y p t o p h a n emission and the retinol absorption band is present for an efficient excitation resonance transfer. The rate constant of energy transfer, k, can be calculated by ktrp-~retino

1 =

=

8.7 × 10:3 JK 2 • ~-4 1

o

. ~)trp

"

o-1 rtrp

. R-6

1 ----~-- = 4.4 " 108 S-1

Ttrp

(7)

Ttrp

where J (=1.35.109) is the spectral overlap, ~2 is the orientation factor (2/3 assumed), ~?(1.3598) is the refractive index, and ~bt~p (=0.08) and Tt~p (1.4 ns) are the fluorescence q u a n t u m yield and lifetime in the absence of energy transfer, respectively, rtrp (=0.87 ns) is the lifetime in the presence of energy transfer. R is the distance in .~ between the transition dipoles of t r y p t o p h a n and retinol. The energy transfer efficiency calculated from Fig. 4 was 44%. An apparent distance, R, was calculated to be 34 .~. The low transfer rate constant and large R value suggest t h a t retinol is bound at one of the tryptophans, completely quenching the fluoresence statically and interacting only weakly with the remaining t r y p t o p h a n residues situated away from the binding site. 3. Rotational relaxation time (p) ~-Lactoglobulin is a prolate ellipsoid with the axial ratio of 2 : 1 [51]. Utilizing the pulse-boxcar averaging technique, a single value of p = 21 ns has been obtained, corresponding to the 20 000 dalton m o n o m e r [2,3]. However, their conditions favor the dimer to be predominant. Polarized fluorescence decay of dansyl-labelled protein yielded p = 60 ns and 29.5 ns for the dimer and monomer, respectively [52]. Using Eqn. 5, we obtain p = 14 ns at 10 MHz and 4 ns at 30 MHz modulation at pH 7.5 (Table III). These values are considerably smaller than the value obtained by dansyl probe [52]. We have also calculated p values for dansyl-labelled ~-lactoglobulin using Eqn. 5, and results are in agreement with [ 52]. Since our dynamic depolarization m e t h o d yields p values TABLE

III

THE ROTATIONAL RELAXATION FLUORIMETRY USING DIFFERENT pH

TIMES OF PROBES

~-LACTOGLOBULIN

AS

p (ns) 10 MHz

30 MHz

fl-Lact o g l o b u l i n - r e t i n o l c o m p l e x 2,0 7.5

14.9 13.7 *

3.1 4.0 *

~-Lactoglobulin-dansyl complex 2,95 7,5

21.4 54.5

21.0 (29.5) ** 31.7 (60.0) **

* F r o m tan ~ analysis, these values b e c o m e 1 2 and 3 ns, r e s p e c t i v e l y (see t e x t ) . ** Taken from [52].

MEASURED

BY PHASE

39

consistent with the pulse method in the case of the dansyl complex, the discrepancy in p values for ~-lactoglobulin-retinol and dansyl complexes is thus puzzling. The fluorescence lifetimes of both probes are certainly long enough for rotational relaxation time measurements. One of several possible explanations is that retinol is located in a segmentally flexible region of the protein, while dansyl binds at a different site. Another is that the probe transition dipole axes of the t w o probes are aligned differently with respect to the protein rotation axes, resulting in differential weighting of one c o m p o n e n t or the other. Weber [53] has, however, recently discussed a method whereby anisotropic rotations can be evaluated utilizing phase fluorimetry. This is briefly discussed below. From Eqn. 5, the maximum value of tan A, (tan A)max , m a y be determined b y equating the coefficient of the 2a' term to the last term raised to the 1/2 power, giving (tan A ) ~ x

Po ¢ ~ F [1 + [1 --P2o)(1 +

(.~2T2)]1/2]

(8)

This maximum value of tan A is reached at a value of the rotational rate given by [(1 -- P~o)(1 + O~2T~)] 1/2 (3 - - P o )

(9)

Weber then described a simple test for the existence of anisotropic rotations; that being the failure of the experimentally determined value of tan A reaching the (tan A)max in plots of tan A vs. temperature. One of these plots is shown in Fig. 7 for the complex as a function of temperature. The horizontal lines labelled 0.2919 and 0.1376 are the calculated (tan A)max at 30 and 10 MHz, respectively. As can be seen, there is a tangent defect, indicating anistotropic rotations. From the quadratic dependence of p on tan A, t w o values for p at

OJ(

0.2919

0.1, OJ2

\

<3 ~" 0.10

)M:

o.08

%,

o.06

3'o3 313

323

Temperoture, K Fig. 7. A p l o t of t a n A vs. t e m p e r a t u r e f o r t h e ~ - l a c t o g l o b u l i n - r e t i n o l c o m p l e x . H o r i z o n t a l l i n e s d e n o t e t h e ( t a n A ) m a x a t e a c h f r e q u e n c y a n d vertical l i n e s are error bars, d e n o t i n g a s t a n d a r d d e v i a t i o n o f t h e measurement.

40 each frequency can be obtained. The choice of the proper root is made either from inspection or by comparison with (tan A)max ; the point at which the roots are at their m a x i m u m and equal in value. In this case, the proper values are 12 and 3 ns with the other roots (49 and 59 ns) being greater than the (tan A)max. This is somewhat curious, however, as the values we eliminated are closer to the dansyl data (Table III) and the expected molecular volume of the fl-lactoglobulin dimer. This question must be examined further. From steady-state measurements and the Perrin equation [29], the rotational relaxation time m a y also be evaluated, approx. 33 ns, which may be taken as the upper limit for the rotational relaxation time for the ~-lactoglobulin-retinol complex. Our data suggest that retinol is bound at a site different from that of dansyl and the retinol site is at a location that is flexible with respect to the bulk of the protein dimer. This accounts for the short relaxation time observed. The fact that the values at two different modulation frequencies are not in agreement further signify anisotropic rotations and in fact indicate that there is a c o m p o n e n t larger than 12 ns and one smaller than 3 ns, the magnitude of their splitting being related to the relative weighting of each component. Using the same phase fluorimetry isotropic rotations have been observed for the peridinin-chlorophyll-protein complex [ 28]. The rotational relaxation time for this system was observed to be 33 ns at 10 and 30 MHz (Mr 3 5 0 0 0 ) and the (tan A)max agreed within a few percent of the experimentally observed value. This very clearly indicates the validity of the technique. In a situation where the rotational motions of the probe are limited to a nonzero anisotropy value (r.) at times long compared to TF [54,55], the limiting anisotropy is related to the average angle (¢r) to which the torsional motions are restricted ro _ 2 r . (3 cos 0 r - - l )

(10)

thereby allowing an evaluation of the average angle through which the chromophore rotates at times long compared to ~F. From our above calculations, we obtain a limiting anisotropy value of 0.098, which corresponds to <¢~>-- 44 °. Thus the overall picture that emerges from the differential polarized phase fluorimetry measurements of rotational relaxation is that of a chromophore bound rigidly at a hydrophobic site which is part of a flexible segment of the protein, on the basis of short, anisotropic rotational relaxation times. Furthermore, the rotation is viewed as being somewhat hindered with the average angle described by the restricted torsional motion approx. 44° .

4. fl-Lactoglobulin-retro retinyl complex In order to examine three fractions from the G-100 column (Fig. 6), blue dextran (2 • 106 daltons) was run .through the same column, to determine its void volume, which corresponded with the 'T' peak, indicating that its molecular weight from Fig. 6 (inset) was most likely underestimated. Running only retinol over the column gave 'T' and 'M' peaks, but no D. 'T' therefore corresponds to micellar retinol. 'M' corresponds to 'free' or small oligomeric retinol miceUes.

41 Fig. 1B shows the absorption spectrum of 'D' fraction (Fig. 6), which corresponds to the molecular weight (dimer) of 36 000 (Fig. 6 inset). Fig. 1C also presents the absorption spectrum of hexane extract from 'D', showing a new derivative of retinol. Thus the highly resolved absorption band is due to the modified retinol c h r o m o p h o r e produced during the chromatography. Hemley et al. [44] have recently described the same p h e n o m e n o n in detail using our chromatographic procedure. Hereafter, we designate this retinyl derivative as retro c o m p o u n d . The retro complex shows a highly resolved CD spectrum (Fig. 2C) corresponding to the vibrational resolution seen in the absorption (Fig. 1B) and fluorescence excitation (hE = 545 nm) spectra. The TF value for free retro c o m p o u n d is 1.7 -+ 0.3 ns, which increased to 9.9 -+ 0.3 ns u p o n binding to ~-lactoglobulin (T F for 'D' showed the same lifetime value). What, then, is the chemical identity of the retro c o m p o u n d ? Anhydrovitamin A was ruled o u t on the basis of: (i) absorption spectrum of ~-lactoglobulinanhydrovitamin A complex (kmax (=392, 377, 356 nm) is 20 nm red shifted compared to ~max (= 3 7 1 , 3 5 2 , 3 3 5 nm) for fi-lactoglobulin-retinol); (ii) rE which changes from 2.5 ns (free) to 1.5 ns u p o n complexing ~-lactoglobulin; and (iii) ~ F which is 37 nm red shifted relative to ~ F of fi-lactoglobulin-retinol (cf. Fig. 4). To characterize the u n k n o w n retro c o m p o u n d , the Fourier transform NMR (in C2HC13 and acetone-d6) and mass spectra of ~-ionone, retro retinyl acetate, anhydrovitamin A and all-trans retinol have been compared. The IH-NMR of the retro c o m p o u n d showed a moderate intensity resonance at 3.0 ppm (13-CH3?), a weak signal at 4.1 ppm (H at 14-C) and another weak resonance at 3.3 ppm. The methyl region at 0.5--2.0 ppm was not well resolved. However, from the comparison of the NMR spectra of r e t r o r e t i n y l acetate and anhydrovitamin A, features of the retro fl-ionone ring are conserved in the u n k n o w n retro c o m p o u n d . In the mass spectrum, m / e 285 (M ÷) was found, along with m / e 156, 106 and 92, suggesting that the conjugated chain is unaltered. These data are consistent with the retro structure proposed b y Hemley et al. [44].

Acknowledgements This work was supported by the R o b e r t A. Welch Foundation (D-182) and the National Science Foundation (PCM75-05001). References 1 F u t t e r m a n , S. and Heller, J. (1972) J. Biol. Chem. 247, 5168--5172 2 Georghiou, S. and Churchich, J.E. (1975) Int. J. Q u a n t u m Chem.: Q u a n t u m Biology Symp. 2, 331-337 3 Georghiou, S. and Churchich, J.E. (1975) J. Biol. Chem. 250, 1149--1151 4 Radda, G.K. (1975) in Methods in Membrane Biol. (Korn, E.D., ed.), Vol. IV, p. 97, P l e n u m , N e w York 5 Sklar, L.A., Hudson, B.S. and Simord, R.D. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1 6 4 9 - - 1 6 5 3 6 Sklar, L.A., Hudson, B.S. and Simoni, R.D. (1977) Biochemistry 16, 5 1 0 0 - 5 1 0 8 7 Piez, K.A., Davie, E.W., Folk, J.E. and Gladner, J.A. (1961) J. Biol. Chem. 236, 2912--2916

42

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