Bioc6imica et Biophysica Acta, 1044 (1990) 361-367

361

Elsevier BBALIP 53408

T h e b i n d i n g of p a l m i t o y l - C o A to b o v i n e s e r u m albumin, Ernest W. Richards

1,2 M i c h a e l

W. Hamm

2 John

E. F l e t c h e r

3 and David

A . O t t o t+~

J Department of Research, The Baptist Medical Cent,_'rs, Birmingham. A L 2 Graduate Program in Nutrition, Rutgers, The State Uni~ersity of New Jersey, N e w B r u ~ i c l ~ N J and t National institutes of Heulttg Division+ of Computer Research and Technology. Bethesa~ M D ( U+S.A+)

(Re¢¢ivocl 11 D e . t u b e r 1989)

Key words: PalmitoyI-CoA; Bovine serum albumin; Fluorescence; 16-(9-Anthroyloxy)palmitos-I-CoA; Anthracene

Bovine serum albumin (lISA) is routindy utilized in vitro to prevent the adverse detergent effects of long-chain acyi-CoA esters (i.e., palmitoyI-CoA) in enzyme assays. Determination of subslrate s~tm-ation kinetics in the presence o f albumin would only be valid if the relationship between bound and free substrate conc~nlrations was known~ T o elucidate the relationship between b o u n d and free imlmitoyl-CoA ¢oneentralions in t h e p c ~ n e e o f lISA, several different teclmiqu~s including ~luilil~rium dialysis, equilibcium partitioning, f l u o r e s ~ n e e polarization a n d direct fluorescence e n h a n c e m e n t were investigated. Direct fluocescence e n h a n c e m e n t using a c u s t o m synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyI-CoA (AP-CoA), was the best approach to this question. M ~ t of t h e relationship between tool of p~dmitoyi-CoA b o u n d per tool of B S A (~) versus - l o g [ f r e e palmitoyI-CoAI revealed that t h e binding of palmitoyI-CoA to l i S A , like palmitate was nonlinear, suggesting t h e presence of m o r e t h a n o n e d a s s of acyI-CoA binding sites. C o m p u t e r analyses of the binding data gave a best fit to the 2,4 two-class S c a t c h a r d model, suggesting the pcesence of two higb-afCmity pcimary binding sites ( k t -- (1.55 :t: 0.46) - 10 -6 M - t ) and f o u r lower affinity secondary binding sites (k2-~ (1.90 -1- 0 . 0 9 ) - 1 0 -8 M - I ) . Fmrther analyse~ using the six p a r a m e t e r stoichi(,metric (stepwise) ligand binding model supports the. e x i s t e n c e of six binding sites with t h e higher affinities r a t e d with the binding of t h e first m o l e o f l~dmitoyl-CoA and w e a k e r binding occurring Mter t h e first two sites are occupied. T h e association constants f r o m this m o d e l of multiple binding diminish sequentially (Le.,K t > K 2 > K 3 > . . . > / K 6), s u g g e s t i r ~ that each tool of long-chain acyl-CoA binds to B S A with decreasing affinities.

Introduction T h e r e are n u m e r o u s reports in the literature regarding the b i n d i n g o f long-chain fatty acids po BSA as well as to h u m a n s e r u m a l b u m i n [1-5]. Collectively. these studies indicate that the free ( u n b o u n d ) fatty acid conc e n t r a t i o n is directly d e p e n d e n t o n the m o l a r ratios o f fatty acid t,3 a l b u m i n . These studies also suggest that t h e b i n d i n g o f long-chain fatty acid to a l b u m i n is a m u l t i p l e site reaction with several b i n d i n g sites of varying affinities w h i c h d e m o n s t r a t e negative cooperativity in binding. F o r this reason, the relationship of free ( u n b o u n d ) to to~a, ( u n b o u n d plus b o u n d ) long-chain fatty acid is no~ linearly related w h e n total c o n c e n t r a Abbreviations: BSA, bovine serum albumin; CPT, earnitine palmitoyltransf,.rase (EC 2.3.1.21); AP-CoA, 16-(9+anthroyloxy)palmitoyI-CoA; D ' r ' r , dithiothreiiol; EGTA, [ethylenebis(oxyethylenenitrilo)ltetracetic acid. Correspondence: E.W. Richards, BaFtist Medical Centers, Department of Research, 701 Princeton Avenue, Birmingham, A L 35211, U.S.A+

tion is varied while m a i n t a i n i n g a c o n s t a n t albunfir. c o n c e n t r a t i o n . However, d a t a regarding the b i n d i n g of activated long-chain fatty acids ,qong-chain a c y l - C o A esters) by a l b u m i n is lacking. A l b u m i n is often used in assays to prevent the detergent effects of long-chain acyI-CoA miceile f o r m a t i o n . These adverse effects have been r e p o r t e d in n u m e r o u s studies investigating the kinetic characteristics of palm i t o y I - C o A utilizing enzymes, particularly c a r n i t i n e patmitoyltransferase ( C l r O of liver m i t o c h o n d r i a [6,7]. F u r t h e r m o r e , several studies investigating the kinetic behavior of C P T atilizecl c o n s t a n t c o n c e n t r a t i o n s o f BSA while v~rying the c o n c e n t r a t i o n of long-chain acylC o A [8-10]. T h e o b s e r v e d kinetic p a r a m e t e r s ( a p p a r e n t Kin) were therefore based o n total a c y l - C o A c o n c e n t r a tions, rath¢r than o n the actual free acyl-CoA concentrations present in the assay system. Additionally, the kinetic behavior of this e n z y m e appears to be dep e n d e n t u p o n the assay c o n d i t i o n s e m p l o y e d [11,12]. This is p e r h a p s d u e to t h e effects of variable assay c o n d i t i o n s on acyl-CoA b i n d i n g to BSA a n d the subseq u e n t alterations in substrate availability.

0005-2760/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

362 Recent reports confirm the importance of considering this binding relationship when c o n d u c t i n g substrate saturation kinetic studies. Bartlett et al. [13] have questioned the validity of conducting kinetic investigations o f m e m b r a n e - b o u n d enzymes which utilize long-chain acyl-CoA esters as substrates in the presence of albumin. In addition, Pauly and McMillin [14] have shown that palmitoyl-CoA binding to albumin contributes to !he observed sigmoidal kinetic profile of the outer mitochondrial CPT. The purpose of this study was to define m o r e exactly this binding relationship by providing m e a s u r e m e n t s of the n u m b e r and affinity of binaing sites on BSA for palmitoyl-CoA. The significance of the present study is related to the routine use of albumin as a substrate binder in assays which utilize long-chain acyl-CoA esters as substrates. An understanding of the interactions between acyI-CoA substrates a n d albumin is therefore essential for the accurate determination o f enzyme kinetics. Materials and M e t h o d s Materials

Palmitoyl-CoA, Tris-HCI a n d dithiothreitol ( D T T ) were purchased from Sigma (St. Louis, MO). 16-(9-anthroyloxy)palmitoyl-CoA was custom synthesized by Molecular Probes (Eugene, OR). Sucrose, potassium chloride and [ethylenebis(oxyethylenenitrilo)]tetracetic acid ( E G T A ) were purchased from Fisher Scientific (Fair Lawn, N J). Clinical reagent g r a d e bovine serum albumin (CRG-7) from A r m o u r (Kankakee, IL) was utilized in all studies. This is a lyophilized, d e f a t t e d albumin preparation that has been subject ".A to several purification procedures including ion-exchange dialysis a n d ultrafiltration to remove any free peptide con.taminat;on. Total fatty acid c o n c e n t r a t i o n was < 0.11 m g / g or < 0.01 • of the albumin dry weight and m o n o meric purity of this BSA preparation was > 90%. In addition, the m a n u f a c t u r e r of C R G - 7 BSA has shown that proteinase activity (based on a Casein assay) was below detectable', limits a n d therefore not present in this albumin preparation. M e t h o d t , 'ogi~ , ~'.: : e s t i g a t e d

A t t e m p t s to develop a technique for investigating the binding o f palmitoyl-CoA to BS-',. were extensive. Although the following techniques wz:e not f o u n d to be quantitatively accurate in the analysis c,f palmitoyl-CoA binding to albumin, they are described, along with their problems, in the following paragraphs. Equilibrium dialysis [15] using S p e c t r a / P o r 2 cl.~alys~ tubing with a molecular weight c u t - o f f of 12 000-14 0G0, was found to be quantitatively inaccurate d u e to the restricted passage of long-chain acyI-CoA through the cellulose dialysis ~ ¢ m b r a n e s . This observation was also

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F i g 1. F l u o r e s c e n c e e x c i t a t i o n - e m i s s i o n s p e c t r a o f A P - C o A . T h e c u v e t t e c o n t a i n e d 10 ~ M A P - C o A in the p r e s e n c e o f 5 0 / t M BSA i n a total v o l u m e o f 3.0 ml o f the p r e v i o u s l y d e s c r i b e d a s s a y cocktail at 3 7 ° C a n d p H = 7.4 ( s e e M a t e r i a l s a n d M e t h o d s ) . E m i s s i o n w a s a t 500 n m tot e x c i t a t i o n s p e c t r a a n d e x c i t a t i o n w a s at 383 n m f o r e m i s s i o n s p e c t r a . Inset: 1 6 - ( 9 - a m h r o y l o x y ) p a [ m i t o y I - C o A .

made in studies investigating the binding of long-chain fatty acids to a l b u m i n [5]. T h e technique o f equilibrium partitioning [2] with modifications [3] was also investigated. Preliminary studies designed to d e t e r m i n e the time necessary to achieve equilibrium between the organic n - h e p t a n e a n d aqueous phases revealed that, a l t h o u g h a significant a m o u n t of the radioactive tracer ([1-14C]palmitoyl-CoA) did partition into the organic phase, equilibrium between the two phases was still not a p p a r e n t after 32 h o f incubation at 37 ° C. S u b s e q u e n t thin-layer c h r o m a t o graphic analysis of the n - h e p t a n e p h a s e indicated that the radioactivity present was in the f o r m of palmitate, rather than palmitoyl-CoA, suggesting a slow hydrolysis of the initial [1-14Clpalmitoyl-CoA a d d e d to the partitioning sys*em. This observation indicates that palmitoyl-CoA has a limited solubility in the organic b u f f e r phase, m a k i n g this t e c h n i q u e i n a p p r o p r i a t e for the analysis o f palmitoyl-CoA binding to BSA. Based on the a b o v e findings, it was deci,'ed that a technique such as fluorescence polarization [16], with the a d v a n t a g e of not having to physically separate b o u n a and free ligand, w o u l d be m o r e useful in the d e t e r m i n a tion o f p a l m i t o y l - C o A b i n d i n g to BSA. P r e l i m i n a r y analysis of a custom synthesized fluorescent p r o b e designed for these studies, A P - C o A (Fig. 1), revealed that there was a m a r k e d increase in the q u a n t u m yield of the b o u n d verses free forms o f the fluorescent t~robe. H o w ever, the actual observed c h a n g e s ia fluorescent anisotropy+ w h e n investigating i n c r e a s i nor c o n c e n t r a t i o n s o f the p r o b e at a fixed macromolecul.e (BSA) c o n c e n t r a tion, were not sensitive e n o u g h to detect changes in the free a n d b o u n d f l u o r o p h o r e concentrations. T h e successful use of fluorescent e n h a n c e m e n t in the evaluation ol d~.e binding o f fluorescenfly labeled probes to mr~cromolecules such as albumin is well d o c u m e n t e d

363 [16,23,24,26]. W e t h e r e f o r e utilized the significant c h a n g e s in f l u o r e s c e n c e q u a n t u m yield as d e s c r i b e d a b o v e to investigate the b i n d i n g o f p a l m i t o y l - C o A to BSA. T h e s e studies are descz;b,.d in detail in the following sections.

t i t r a t i e n s had he.en m a d e . A f t e r each addition, the samp l e s w e r e a l l o w e d to e q u i l i b r a t e for 5 rain a n d the f l u o r e s c e n c e intensities w e r e m e a s u r e d in triplicate.

Computer analyzis of binding Fluorescence binding studies All s t u d i e s o f p a l m i t o y l - C o A b i n d i n g t o BSA were c o n d u c t e d at 3 7 ° C in a 3 ml a s s a y s y s t e m ( p H 7.4) c o n t a i n i n g 200 m M sucrose, 40 m M KC1, 10 m m TrisH C I ( p H 7.4), 1 m M E G T A a n d 0.5 m M D T T , with B S A a n d p a l m i t , ' y ! - C o A c o n c e n t r a t i o n s as indicated. This m e d i u m w a s u s e d b y o t h e r s in t h e kinetic investig a t i o n o f t h e o u t e r m i t o c h o n d r i a l C P T [10]. F l u o r e s c e n t intensities w e r e m e a s u r e d in an S L M 4800 spectrophoto-fluorometer of the L-format (SLM, C h a m p a i g n , I L ) at the i n d i c a t e d p r o t e i n c o n c e n t r a t i o n s . T e m p e r a t u r e s in the f l u o r e s c e n c e c u v e t t e well o f the f l u o r o m e t e r w e r e m a i n t a i n e d at 37 o C. All b i n d i n g d a t a w e r e o b t a i n e d at an e x c i t a t i o n w a v e l e n g t h o f 383 n m a n d a n e m i s s i o n w a v e l e n g t h o f 4 4 2 rim, which w e r e d e t e r m i n e d f r o m p r e l i m i n a r y spectral analysis o f A P C o A in the p r e s e n c e o f a s a t u r a t i n g c o n c e n t r a t i o n o f B S A ( 5 0 / ~ M ) (Fig. 1). T h e inner filter effect w a s minimixed b y k e e p i n g the a b s o r b a n c e o f all p r o b e c o n c e n t r a t i o n s u s e d in t h e s e studies b e l o w 0.05 a b s o r b a n c e units. E x c i t a t i o n a n d e m i s s i o n b a n d w i d t h s w e r e r o u t i n e ly l u t e d at 8.0 a n d 2.0 n m , respectively. A 1 m M s t o c k s o l u t i o n c o n t a i n i n g 80% p a l m i t o y l C o A a n d 20% A P - C o A w a s t h e n a d d e d to a c h i e v e the desired total palmitoyl-CoA concentrations (palmitoylCoA + AP-CoA). The assay cocktail was preincubated in a 3 7 ° C w a t e r b a t h w i t h t h e r e s p e c t i v e a l b u m i n c o n c e n t r a t i o n s in P r o s i l - t r e a t e d p o l y s t y r e n e test t u b e s ( t o p r e v e n t n o n s p e c i f i c ligand b i n d i n g to the test tubes). T u b ~ , w e r e v o r t e x e d a n d the c o n t e n t s w e r e t r a n s f e r r e d to 3-ml q u a r t z cuvettes, w h i c h w e r e p l a c e d in the fluor o m e t e r for a 3 rain t e m p e r a t u r e e q m l i b r a t i o n p e r i o d . T h e f l u o r e s c e n t i n t e n s i t y w a s m e a s u r e d in triplicate.

Competition studies T h e p r e s e n c e o f the f l u o r e s c e n t a n t h r a c e n e m o i e t y o n A P - C o A suggests that t h e b i n d i n g a f f i n i t y a n d / o r sites f o r a l b u m i n m a y d i f f e r f r o m that o f p a l m i t o y l - C o A . In o r d e r to e v a l u a t e p o t e n t i a l d i f f e r e n c e s , we c o n d u c t e d d e t a i l e d c o m o e t i t i o n s t u d i e s to investigate t h e b i n d i n g affi,tity o f a l b u m i n for A P - C o A v e r s u s p a l m i t o y I - C o A . In t h e s e studies, t h e f l u o r e s c e n c e intensities o f s a m p l e s c o n t a i n i n g either 1 0 / ~ M , 20 ~ M o r 4 0 / t M A P - C o A , in the p r e s e n c e o f 1 9 . 4 / t M BSA, w a s m e a s u r e d u n d e r th,. s a m e a s s a y c o n d i t i o n s as d e s c r i b e d p r e v i o u s l y . T h e samp l e s w e r e t i t r a t e d w i t h either 2-/tl a d d i t i o n s o f 6 m M o r 6-/tl a d d i t i o n s o f I m M u n l a b e l e d p a l m i t o y l - C o A ( 4 / t M o r 2 / t M i n c r e a s e s in total p a l m i t o y l - C o A c o n c e n t r a tion, respectively). In e a c h case, the t o t a l v o l u m e o f the a s s a y s y s t e m w a s c h a n g e d b y less t h a n 0.05% a f t e r all

T h e d a t a o b t a i n e d f r o m the f l u o r e s c e n c e e n h a n c e m e n t studies were fitted to m a t h e m a t i c a l n,o.tel~ Oy m e a n s o f an interactive c o m p u t e r p r e p a r e , ?,4L.xb [3,17-20]. T h e c o m p u t e r u.,ed w a s the D E C K L - 1 0 d u a l process, or s y s t e m as c o n f i g u r e d at the Division o f C o m p a t e r R e s e a r c h a n d T e c h n o l o g y , N a t i o n a l Institutes o f H e a l t h a n d w a s a c c e s s e d t h r o u g h r e m o t e terminal with v i d e o ( g r a p h i c s ) display. T h e p a r t i c u l a r t e r m i n a l u s e d in these a n a l y s e s w a s t h e T e k t r o n i x 4025. Results

Competition szudies A r e p r e s e n t a t i v e c o m p e t i t i o n s t u d y is p r e s e n t e d in Fig. 2. T h e results f r o m t h e s e s t u d i e s d e m o n s t r a t e d a r e d u c t i o n in t h e f l u o r e s c e n c e i n t e n s i t y f o l l o w i n g the iltcrease in t h e p r o p o r t i o n o f n o n - f l u o r e s c e n t l y l a b e l e d palmitoyl-CoA, indicating AP-CoA and palmitoyI-CoA c o m p e t e for the s a m e sites o n albu.nin. T h e s e findings suggest, even w i t h the d i s t i n c t d i f f e r e n c e s in s t r u c t u r a l c h a r a c t e r i s t i c s o f t h e p r o b e ( A P - C o A ) versus palmitoyl-CoA, that the observed relationship between APC o A a n d B S A is r e p r e s e n t a t i v e o f the r e l a t i o n s h i p bet w e e n p a l m i t o y l - C o A a n d BSA.

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Fig. 2. Competition study of AP-CoA titrated with unlabeled palmitoyI-Cx~A in the presence of 19.4 tam BSA. The fluorescence intensity of 3+0 ml samples contahting either 20 laM, (O), or 40 pM, (e). AP-CoA in the presence of 19.4 taM BSA under the previously described assay conditions at 37°C were measured (Ex 383 nm, Em 442 nm). These samples were then titrated with 2 pl add/tions of 6 mM unlabeled palmitoyI-CoA (4 t~M incremental increases in unlabeled palmitoyI-CoA), allowed to equilibrate for 5 rain and measured for fluorescence intensity.

364

Fluorescence characteristics mitoyl-CoA

of 16-(9-anthroyloxy)pal-

Fig. 3 presents the fluorescence emission spectra ~f the probe (AP-CoA) in the presence a,id absence of BSA. The fluorescence intensity of the probe (10/~M) in the aqueous assay cocktail alone was not significant ( - B S A curve), however, when albumin (50 /~M) was a d d e d to the assay cocktail ( + BSA curve), the intensity was greatly enhanced, with a 22 nn= shift in wavelength of the emission maxima. This quantitatively significant change in fluorescence intensity as a result o f binding to albumin was utilized as an indicator of the binding in teraetions. The areas u n d e r the emission curves o f Fig. 3 were directly p,oportionai to the q u a n t u m yield of AP-CoA. Q u a n t u m yield determinations were m a d e using a modification of the m e t h o d of W a r d a n d Cormier [21] with quinine sulfate as a reference standard. T h e curves of Fig. 3 revealed that the q u a n t u m yield (0.058) o f the probe in the absence of albumin increased approx. 12-fold to 0.71 when albumin was a d d e d to the aqueous assay cocktail. These changes are d u e to alterations in the e n v i r o n m e n t of the fluorescent probe a n d provide strong evidence of the binding between the probe and albumin. Weber and Lawrence [22] described that while only weak fluorescence is emitted when h y d r o p h o b i c probes are dissolved in aqueous solutions, intense fluorescence occurs when such probes are b o u n d to BSA or when they are placed in an organic solvent.

16-(9-Anthroyloxy)palmitoyl-CoA binding to BSA The relative fluorescence intensities of solutions containing increasing concentrations of 80%/20% palm i t o y l - C o A / A P - C o A a: 0 / t M , 19.4 tam a n d 300 taM BSA are presented in Fig. 4. F u r t h e r increases in BSA

.

300

400 500 600 Emission Wavelet~th (,am)

700

Fig. 3 . E m i s s i o n s p e c t r a o f A P - C o A in t h e p r e s e n c e a n d a b s e n c e o f BSA. T h e c u v e t t e s 6 o n t a i q e d 10 t i M A P - C o A i n t h e p r e s e n c e a n d a b s e n c e o f 50 t t M B S A in 8 t o t a l v o l u m e o f 3.0 m l o f the p r e v i o u s l y d e s c r i b e d a s s a y cocktail a t 37 ° C a n d p H = 7.4. E x c i t a t i o n was a t 383 d m as d e t e r m i n e d f r o m Fig- 1.

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[PC-AP] (pM) Fig. 4. Plots o f r e l s t ; v e f l u o r e s c e n c e i n t e n s i t i e s as a f a o c t i o ~ of t o t a l 8 0 ~ :20¢g p a l m i t o y i - C o A : A P - C o A c o n c e n t r a t i o n at c o n s t a n t B S A c o n c e n t r a t i o n s . C u r v e A. [BSA] --- 300 txM; c u r v e B [BSA! = 19.4 p M ; c u r v e C, [BSA] = 0 p M .

c o n c e n t r a t i o n above 300 # M resulted in n o f u r t h e r e n h a n c e m e n t of fluorescence intensity o r q u a n t u m efficiency, indicating that p a l m i t o y l - C o A was m a x i m a l l y b o u n d at this protein c o n c e n t r a t i o n (results not shown). in addition, the relative fluorescence intensities o f solutions containing increasing c o n c e n t r a t i o n s of 100~; A P C o A in the presence o f 25 taM or 50 ~tM a l b u m i n revealed an overlapping linear increase in fluorescence yield. This indicates that the fluorescence yield of A P CoA is i n d e p e n d e n t of the degree o f saturation a n d validates the use o f a two-state e q u a t i o n relating the fluorescence changes to the fraction o f probe b o u n d . F u r t h e r m o r e , since a l b u m i n is such a strong b i n d e r o f palmitoyl-CoA a n d AP-CoA, the fluorescent b i n d i n g studies presented in this work did n o t result in free palmitoyl-CoA c o n c e n t r a t i o n s high e n o u g h to p r o d u c e micelles [23,24]. T h e lower curve (C) shows the titration ff the fluorescent p r o b e into the assay cocktail in the a b s e n c e o f albumin and therefore gives the values of free p r o b e fluorescence as a f u n c t i o n of p r o b e concentration. T h e u p p e r curve (A) represents the titration of the p r o b e into the assay cocktail in the p r e s e n c e of 300 /~M albumin, a protein c o n c e n t r a t i o n at which essentially all of the p r o b e is b o u n d . C u r v e B represents the fluorescence intensities o f p a l m i t o y l - C o A in the presence o f 19.4 /xM (0.13%) BSA. These fluorescence h,'tensities can be regarded as arising f r o m the b o u n d pal.,nitoyiC o A c o n c e n t r a t i o n giving the s a m e observed intensity of curve A. The fluorescence intensities o f free a n d b o u n d p r o b e (0 /tM BSA a n d 300 taM BSA, respectively), as well as the observed fluorescence at the lower protein c-~ncentration ( 1 9 . 4 / t M ) , are used to calculate X, the fraction o f the p r o b e bound.

365 "l'he fraction of probe b o u n d ( X ) to BSA was calcuIated from the following equation [25,26]: ( lo/xf ) -

X

(.'b,%)-

6

1 z

where 1o a n d I t are the fluorescence intensities of a given probe c o n c e n t r a t i o n in the presence o f a low BSA c o n c e n t r a t i o n (19.4 # M ) and in !he absence of BSA, respectively, lb refers to the fluorescence intensity of the same total p r o b e c o n c e n t r a t i o n in the presence of a saturating BSA c o n c e n t r a t i o n ( 3 0 0 / t M ) . T h e average n u m b e r o f p r o b e molecules b o u n d per mol o f albumin (~) was calculated for points along the titration curve using the following formula:

3

0 ~,=

X( A,/P,)

7

6

5

4

where A t is total p r o b e c o n c e n t r a t i o n ( # M ) and Pt the total a l b u m i n conceW.ration ( # M ) [25]. Computer analysis o f binding data T h e binding d a t a were entered into the M L A B program (described in Materials a n d Methods) in the form of two-dimensional arrays of n u m b e r s representing b o u n d concentrations versus free c o n c e n t r a t i o n s for the binding of A P - C o A to BSA, These d a t a were derived f r o m m e a s u r e m e n t s of total (controlled) a n d b o u n d (measured) m o l a r concentrations, at four different molar c o n c e n t r a t i o n s of BSA, as described previously. The d a t a were then c o m b i n e d into a single d a t a set with the free c o n c e n t r a t i o n ( c ) a s the i n d e p e n d e n t variable, and tool b o u n d per tool of BSA (~), as the d e p e n d e n t variable. Using the M L A B least-squares fitting al-

3

2

Fig. 6. Log plot o f A P - C o A b i n d i n g to BSA. BSA c o n c e n t r a t i o r t s were 1 . 0 p M ( 0 ) . 2.5 FtM ( o ) , 12.9 # M ( + ) a n d 19.4 /xM (,~) in t h e a s s a y c o c k t a i l at 3 7 " : C a n d p H = 7.4. E x c i t a t i o n w a s a t 383 a m a n d e m i s s i o n ~a~ at 442 n m . T h e f o r m u l a for t h e c u r v e was: ), = 20.063 + 5.5127x

+ 0.39909x

2, r 2 ~ 0 . 9 8 0 .

gorithm [17], these d a t a were fitted to the free parameter Scatchard form a n d are presented in Fig. 5 where (~) is the molar ratio of b o u n d palmitoyl-CoA to alb u m i n a n d [Free] is the c o n c e n t r a t i o n of u n b o u n d palmitoyl-CoA. T h e log representation of the binding data a n d the c,~,'responding curve are presented in Fig. 6. As was theoretically expected, sincu the p a r a m e t e r s of the binding equation are (b) and (c), and not protcir, concentration° no differences in p a l m i t o y l - C o A binding to BSA were evident over the range of a l b u m i n cor, centrations employed (1.0-19.4/tM). F r o m the resulting derived parameters, a two-class Scatchard model was determined. T h e best fitting models were the 2,4 and 2,5 models, suggesting the presence of two strong (high) affinity sites in the p r i m a r y class a n d four or five sites in the s e c o n d a r y class. The C_'rived parameters are presented in Table I. Since the Z 4 ran Jet

TABLE

!

Summary o/ binding parameters for 16-(9-anthroyloxy)palmttoyl C~A binding to bovine ~erum albwnin

1

P a r a m e t e r s a r e m e a n s ± S .E.

Binding parameters

o

3

L o q [ F r e e ] (M)

0

1

2

3

4

5

6

: [Free]

Fig. 5. S c a t c h a r d p l o t o f A P - C o A b i n d i n g t o BSA. B S A c o n c e n t r a t i o n s w e r e 1.0 p M (13), 2.5 # M ( o ) , 1 2 . 9 / t M ( + ~ a n d 1 9 . 4 / t M (,~) in t h e a s s a y c o c k t a i l a t 3 7 ° C a n d p H = 7.4, E x c i t a t i o n w a s at 383 n m a n d e m i s s i o n was at 442 n m . T h e e x p o n e n t i a l f o r m u l a for t h e c u r v e was: Y --- 5.4280- 10 ( ~ 0 . 4 9 5 o ~ r 2 ,~ 0.934.

( H i g h - a f f i n i t y silos) kt ( L o w - a f f i m t y sites) k 2 Sum of squares

Scatchard modal (high-, l o w - a f f i m t y sites) 2,4

2,5

1.55 ± 0.46 .10-6 M-1 1.90 21:0.09 .10-s M-t 2.01587

1.65 =l:0.47 .10-6 M-! 1.30 ,.t-0.01 .10-s M-I 2.11792

366 T A B L E 11 Stepwise

binding parameters

for

l&-(9-anthro)'loxy)t~almttoyl-CoA

bind-

i n g to b o v i n e s e r u m a l b u m ~ n

a K t = 3 . 1 8 . 1 0 -6 M -1 " K 2 = 0 . 8 2 . 1 0 - 6 M -n b K j = 0.58.10 - s M - i bK4__0.33.10-s M-1 bKs=0.17.10-SM -n bK6=0.12.10-gM-I • H i g h - a f f i n i t y b i n d i n g sites. t, L o w - a f f i n i t y b i n d i n g sites.

has a slightly better sum of squares (Table I), it was chosen as the best fitting model. For the sake o f comparison, the six parameter stoichiometric (stepwise) model [27] was also applied to the d a t a obtained from the fluorescence e n h a n c e m e n t studies. This model is based on the following formula: (~)

Kzc + 2KIK2c

2 + . . . + 6 K n ... K ~ c ~

1 + K1c + K11~2c 2 + ... + K t ... K6c 6 *

where K, is the individual association c o n s t a n t for each tool of the ligand, palmitoyl-CoA. C o n t r a r y to the Scatchard model, there is no grouping of the individual binding sites into separate binding classes in this model. The six stepwise binding parameters derived from this analysis are presented in Table II. T h e g r a p h of this model overlays that of the 2,4 Scatchard model (results not shown). In an a t t e m p t to evaluate the relationship between b o u n d and free palmitoyl-CoA concentrations, we conducted investigations of palmitoyi-CoA binding to albumin, utilizing the technique o f equilibrium dialysis as described previously. During the course of these studies, several problems concerning the passage of palmitoylCoA across the dialysis m e m b r a n e b e c a m e apparent, rendering the quantitative values obtained from these studies invalid. A l t h o u g h the results were quantitatively invalid, the nonlinear relationship observed between b o u n d and free palmitoyl-CoA concentrations (data not shown) in the presence of albumin, followed a pattern identical :o :hat reported for the free fatty acid palrr,.itate lai. In ~ddition, the results from the fluorescence binding ztu0it::, confi.qn this nonlinear relationship between b o u n d and free palmitoyl-CoA in the presence of albumin, as reflected by the (b) ver--:s --log[Free] curve of ~ig. 6. We also carried o u t stud;es utilizing the technique of equilibrium partitioning [2] ~.i~h modifications [3]. However, the h~sotubility of paln~Jtcyl-CoA in organic solvents prevented the use of this techi~q'~-.

Discussion Previous reports concerning the binding of fluorescent probes to multiple binding sites o n albumin

[1625,26,28] have collectively shown that the use of the fluorescence technique p~esented in this work is valid and provides a m e a n s by which ~igand-macromolecule interactions can be quantitatively d.:termined w i t h o u t the constraints of physical separation of b o u n d a n d free ligand. Several aron,atic probes, which are virtually nonfluorescent in an aqueous solution, b e c o m e strongly fluorescent in n o n a q u e o u s solvents, or ~vhen b o u n d to h y d r o p h o b i c sites in proteins [221. The fluorescent probe, A P - C o A (Fig. 1), possesses this unique characteristic, making it useful for the investigatioit o f p a l m i t o y l - C o A binding to albumin. As clearly shown in Fig. 3, this probe exhibited m a r k e d increases in fluorescence intensity w h e n it was b o u n d to the binding sites of a l b u m i n . Conversely, the p r o b e yielded a relatively low fluorescence intensity when it was in an a q u e o u s e n v i r o n m e n t , such ,~ the assay m e d i u m e m p l o y e d in these studies. This quantitative e n h a n c e m e n t o f fluorescence intensity, u p o n binding to albumin, was utilized as a n indicator o f palmitoyl-CoA binding to albumin. Carefully designed competition studies investigating the affinities of A P - C o A verses p a l m i t o y l - C o A for alb u m i n binding showed a linear decrease in fluorescence intensity that close!y coincided with the decrease in ApoCoA b o u n d to BSA. Although the a n t h r a c e n e moiety of A P - C o A potentia.'ly contributes to the h y d r o p h o b i c interactions between A P - C o A and BSA, the linear decrease in fluorescence yield of the binding c o m p l e x observed with the titration of p a l m i t o y l - C o A indicates competition between the probe ( A P - C o A ) a n d palmitoyl-CoA for the same or adjacent binding sites. These studies provide the first reported insight into the binding relationship between p a l m i t o y l - C o A a n d albumin. F m t h e r suppoxt o f our findings are offered b y the fact that the relationship between b o u n d a n d free palmitoyl-CoA obtained in the fluorescence studies closely parallels the observations o f others [4,27] regarding the binding of long-chain fatty acids to BSA. C o m p u t e r analyses of the results obtained f r o m the binding studies revealed that the d a t a was bes. fitted to the 2,4 Scatchard model, indicating two classes o f binding sites for palmitoyl-CoA (one class consisting of two high-affinity sites, k~ = (1.55 + 0.46)- 10 -6 M - l ; a n d one class consisting of four low-affinity binding sites, k 2 = ( 1 . 9 0 + 0.09)- 10 -g M - I (Table I). A l t h o u g h the 2,4 Scatchard model was chosen as the best fitting model, since it h a d a slightly better sum of squares (Table I), the 2,5 Sc.atchard model was al~,o ~ p a t i b l e with the experimental d a t a ( k I = (1.65:1: 0.47) - 10 - 6 M - ~ ; k 2 -----(1.30 :t: O.01)- 10 - s M -n (Table I)). Constants for the stepwise equilibrium m o d e l were estimated f r o m the 2,4 Scatchard p a r a m e t e r s presented in Table I [20,27]. These estimates were refined by the model-fitting p r o c e d u r e described by Fletcher et al. [27] and K n o t t a n d Shrager [29], which analyzes the d a t a directly in terms of stepwise association constants. These

367 constants clearly indicate that the binding of palmitoyi-CoA to BSA can be described in terms of multiple stepwise equilibria. As is the case for long-chain fatty acid binding to BSA [4], the first association constant, K~, is significantly larger than the remaining association constants (K2-Kt). F u r t h e r m o r e , our data d e m o n s t r a t e that the stepwise association constants diminish sequc.ntially and that each tool of palmitoyl-CoA binds to BSA with a decreasing affinity. T h e results of the analyses of palmitoyI-CoA binding to albumin indicate that the relationship between b o u n d a n d free palmitoyi-CoA c o n c e n t r a t i o n s in the presence o f albumin is not linear (Fig. 5 and 6). As m e n t i o n e d earlier, these result resemble those of Spector et al. [4], investigating the binding of palmitate to BSA by the technique o f equilibrium partitioning. The --Ioglfree substrate] verses molar ratio plot of o u r d a t a (Fig. 6) falls within the same range of the plot for palmitate [4]. In addition, the stepwise equilibrium p a r a m e t e r s observed by Spector et al. [4 ! for palmitate binding to BSA fall within the same range of m a g n i t u d e as our values reported in Tables I and II for palmitoyI-CoA. F u r t h e r support of our findings c o m e from recent work investigating the i m p o r t a n c e of acyl-CoA availability in the interpretation o f substrate saturation kinetics on the outer C P T [14]. In these studies, binding competition between mitochondrial b o u n d paimitoylC o A (representing free substrate c o n c e n t r a t i o n ) a n d a l b u m i n b o u n d palmitoyl-CoA (representing b o u n d substrate c o n c e n t r a t i o n ) were measured. T h e s e data, w h e n fitted to the Scatchard model, indicated the prese n c e o f five or six a l b u m i n binding sites for palmitoylCoA. As indicated above, o u r data, utilizing a totally different technique, confirms the presence of six binding sites for p a l m i t o y i - C o A (Table I a n d II). It is c o n c l u d e d that the binding o f palmitoyl-CoA to BSA is consistent with i n d e p e n d e n t site binding w;.th two p r i m a r y high-affinity sites and four or five seco n d a r y lower affinity sites. While one cannot rule out negative cooperativity in the sequential binding process as responsible for this binding curve, there is no direct evidence to indicate that this is the case. it is clear from this work that one must consider the binding of long-chain acyl-CoAs to BSm when studying substrate saturation kinetics of enzymes utilizing acyiC o A as substratc. Acknowledgements We wish to thank Dr. Barbara Zilinskas for the use o f the SLM 4800 s p e c t r o p h o t o f l u o r o m e t e r a n d Dr. Rashida Karmali for her assistance in these studies. We

also wish to thank Dr. J u d y Stotch for her helpful suggestions throughout these studies. This work was supported in part by New Jersey Agricultural E x p e ~ m e n t Station State Funds, Biomedical Research Support G r a m PHS R R ~7058-20.

References 1 A s h b r o o k . ,I.D., Spector. A.A and Fletcher, J.E. (1972) J. Biol. C h e m . 247, 7038-7042 2 G o o d m a n . DeW.S. (195~) J. A m . C h e m . Soc. 80, 3892-3898, 3 Spector. A.A., John, K- and Fletcher, ,I.E. (1969) J Lipid Res. I0, 56-67. 4 Spector. A,A., Flelcher, ,I.E. a n d A s h b r o o k , .I.D. (1971) Biochemistry 10, 3229-3232~ 5 Spector, A.A. ~I075) J. Lipid Res. 16, 165-179, 6 Bremer, J. and N o r u m , K.R. (1967) J_ Biol. C h e m . 242, 1744-1748. 7 WoldegiorgJs. G-, Bremer, J. and Shrago. E. (1985) Biochim. Biophys. Acta 837. 135-1zh'3. 8 C o o k . G . A ~1984) .i. Biol. C h e m . 259. 12030-12033. 9 H a r a n o . ~t. Kowal, J., Y a m a z a k i . R., Lavine. L. and Miller. M. (1972~ Arch. Biochem. Biophys_ 153, 426-437. 10 Saggerson, E.D. and C a r p e n t e r . C,A. (198t) F E B S Lett. 132. 1646- t 68. 11 Bremer, .i., Woldegiorgis, G., Schafinske. K_ and Shrago, E, ~1985l Biochim. Biophys. Act~ 833, 9 - ! 6 12 Saggerson, E.D. (1982) B i o c h e m J. 202, 397-44-t5 1? Bartlett, K., Bartlett, P., Battiest, N. and Sherratt, H.SIA. ~t985~ B.I Lett. 229, 559-560. 14 Pauly. D.F. and McMillin..I~B. (1988) J. Biol. C h e m . 263, 1816018167. 15 M c C o r r m c k , K. a n d Notar-Francesc, o. V..I_ (1983) Biochem. ,i- 216, 495-498. 16 Bentley, K . L , T h o m p s o n . L.K,. Klebe, R.J. and H o r o ~ l t z , P.M. (1985) B i o T e c h n i q u e s 3, 356 • 365. 17 Fletcher, ,I,E, and Shrager, R.I, (1968) A User's G u i d e to LeastSquares M o d e l Fitting. D C R T Technical R e p o r t No. 1. N a t i o n a l Institutes o f Health. Bethesda, M D . 18 Fletcher. J.E. and Spoctor. A.A. (1'~68) C o m p u t . B i o m e d Res. 2. 16,4-175. 19 Fletcher..I_E. and Spector, A , A (IC,~7) Mol. Pharrn 13, 387-399. 20 Fletcher, .I.E. (1982) T h e Analysis of Equilibrium Binding D a t a by the Fitting of Models, D H H S / P H S / N I H Repor~ G P O 1982-361i 32. 578. 21 Ward, W . W and Cormier. M.,I. (1979).t. B,ol. Chem. 254, 781-788. 22 Weber. G. and Laurence, D . J R _ ~19~4) Bit, chem. J. 56, 3 1 - 3 9 . 23 Constantinides, P.P. and Steim. J.M {1985) J. Biol. Che~_ 260. 7573-7580. 24 Powel, (3.L., G r e t h u s e n . . I . R . . Z i m m e r m a n . J.K., Evans. C.A, and Fish, W.W. (1981) J. Blot, C h e m . 256. 12740~i2747. 25 .tun, H.W., Mayer, R.T., Himel, C.M. and Luzzi, L.A. (I971) J. Pharm. Sci. 60, 1821-1825. 26 Nalk, D.V., Paul, W . L , Threatte. R.M. and Schutman, S.G, (1975) Anal. C h e m . 47, 267-270, 27 Fletcher, ,I.E.. Spcctor, A,A. and A s h ~ r o o k . . i . D . (1971)) Bi,~chemistry 9, 4580-4587, 28 Ma. J.K.H., Jun. H.W. and Luz-i. L.A. (1q73) I. Pharm. S c i 62, 2038-2040. 29 K n o t t , G. and Shrager, R.I, (1972) C o m p u t e r Graphic~ 6, 138-151.

The binding of palmitoyl-CoA to bovine serum albumin.

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA)...
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