BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL ABSORPTION AND FLUORESCENCE SPECTRA OF POLYENE ANTIBIOTICS IN THE PRESENCE OF HUMAN SERUM ALBUMIN

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1 Vol. 44, No. 3, March 1998 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL Pages ABSORPTON AND FLUORESCENCE SPECTRA OF POLYENE ANTBOTCS N THE PRESENCE OF HUMAN SERUM ALBUMN Dana Romann, Beatrz Farrugga and Gullermo Pc6 Departamento de Qumca-Fsca and nsttuto de Fsologa Expermental (CONCET). Facultad de Ceneas Boqumeas y Farmaeeutcas. Unversdad Naconal de Rosaro. Supacha 57 (2) Rosaro. Argentna Receved September 26, 1997 ABSTRACT The alteraton n the fluorescence spectra observed for the polyene antbotcs: nystat_, and amphotercn B n the presence of human serum albumn s due to a decrease n the polar character of the antbotc envronment when these are bound to the proten. Amphotercn B showed two types of bndng stes, the frst havng very hgh affnty ( M a) and a secondary bndng ste wth an affnty one order lower than the prmary stes. Ths secondary bndng ste was very senstve to temperature change. Nystatn yelded only one type of bndng stes wth an affnty of M-~. An electrostatc component was found n the bndng of both lgands, as well as an mportant dsorder at the proten bndng stes. However the secondary bndng ste for AMP showed negatve entrope change value, whch suggests dfferent mechanm of bndng respect to the prmary one. Conformatonal change nduced by the temperature n the albumn molecule was detected by nystatn bndng. Fatty acds produced an nterference n the bndng of both antbotcs to albumn. Keywords: albumn; polyene antbotc; nystatn; amphotercn B. NTRODUCTON Optcal probes have provded very useful nformaton about the structure and functon relatonshp n protens. Generally, these probes are polyunsaturated molecules, whch acqure emsson of the fluorescence or modfy ther absorpton spectrum when they are bound to a proten. Prevous reports have shown that polyunsaturated lnear fatty acds such retnoc and cs-parnarc can be used as an optcal probe to detect conformatonal change n proten (1), bologcal membranes and lposomes (2). The polyene antbotcz nystatn and amphotercn B are unsaturated macroldes wth amphpatc nature, whch are used n the therapy of systemc fungal nfectons (3). nstead, nystatn and amphotercn B have been wdely appled as probes for sterol locaton n bologcal membranes because dramatc spectral alteratons of ther fluorescence and absorpton spectra are nduced by formaton of sterol-antbotc complex. These facts and the /98/ / Copyrght by Academc Press Australa, All rghts of reproducton n any form reserved

2 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL proposed pharmacologcal relevance of these polyene antbotcs prompted us to carry out an absorpton and fluorescence study of nystatn and amphotercn B-human serum albumn complexes formaton wth the goal of obtanng nformaton of the molecular mechansm of ths nteracton. MATERALS AND METHODS Reagents: nystatn (NYS), amphotercn B (AMP) and human serum albumn (HSA) fatty acd free (.5 %) were purchased form Sgma Chem. Co (USA) and used wthout further purfcaton. The polyene antbotcs were dssolved n dmethyl sulfoxde and kept n the dark at -2~ no alteraton beng observed n ther spectral propertes for two months. Spectral measurements: fluorescence measurements were recorded n a Jasco FP 77 spectrofluorometer usng lxl cm thermostatzed cuvette. Correcton for nner flter was not necessary because the absorpton of the soluton was less than.1 at the exctaton and the emsson wavelength. Absorpton measurements were recorded on a Beckman 64 spectrophotometer usng a lxl cm thermostatzed cuvette. The polarzaton of the fluorescence was calculated as p =(F // - F ) / (F// + Fz ), beng F// and F the horzontal and vertcal components of the fluorescence Bndng ofpolyene antbotcs to lsa: t was performed by fluorescence spectroscopy, measurng the enhancement of the fluorescence of the lgands when they are bound to the proten, accordng to prevous reports (1). The enthalpc change (AH ~ was calculated form the Van't Hoff equaton: d(lnk)/dt =- AH~ 2, assumng an ndependence of the AH ~ wth the temperature change, the free energy (AG ~ from AG ~ = -RT nk and the entropc change as: AS ~ = (AG ~ AH~ Analyss of the bndng curves: the bndng sotherms were analyzed by a general multparametrc curve-fttng program for non-lneal regresson analyss, usng the equaton for two ndependent classes of bndng stes: r= ~nc/(1 +Klc), where r/c s the bndng rato,.e., the bound lgand concentraton over proten concentraton, n, and ~ are the number of bndng stes and ther correspondng assocaton constant and c s the free lgand concentraton. For cooperatve bndng, the modfed form of the sotherm was used: r= Kn cy / (l+kc v) (4) beng y the cooperatve coeffcent or Hll coeffcent. RESULTS Absorpton spectra of NYS and AMP." Fg. 1 A-B shows the absorpton spectra of the NYS and AMP n the absence and presence of HSA at ncreased concentratons of the proten. From these spectra, the dfference spectra were calculated as shown n Fg. 2 A-B. t can be seen that the dfference spectrum for NYS proved to be very poor, wth two small negatve peaks between 3 nm and 325 nm. Upon addton of HSA, a strong alteraton n the spectrum of AMP was observed (Fg. 2-B). The vbratonal progresson seemed to be affected by decrease of ntensty at 35 nm and two small peaks around the zone of 4 nm. Only the decrease of the absorbance at 35 nm can be consdered as apprecable wthout modfcaton n the peaks poston. 596

3 BOCHEMSTRY and MOLECULAR BOLOGY NTERNATONAL.1.1 w cy O3 s -.1.elL. ~ ~ "" r ' 9 "%'....~.. j k.l" J" l~.. 9 :, 9, m ~1 9 k,,.',%'! r;".,. ;!! 1.4J (,O 123 rk O m ,a,,'.,- r %.. l ~' s.' t)/-',.j WAVELENGTH (nm) WAVELENGHT (nm) Fg. 1: Absorpton spectra of NYS (A) and AMP (B) at 2/~M n the absence and presence of HSA. ( ) only antbotc. Proten concentraton: 5/zM. ( ), 7/zM ( ), 2 p,m. Sodum phosphate buffer medum 1 mm, ph 7.4 Temperature 2~.5.7 LL ro z f C n,- U3 m.4.,3.2.1 _d C,) ry Oq B!.2 /.,..r#r'l. S O. 1,,,,,, f ~,~,! ;,/ % WAVELENGHT (nm) WAVELENGHT (nm) Fg. 2: Dfference absorpton spectra of NYS (A) and AMP (B). The data have been taken from Fg 1. Proten concentraton: 5/~M. ( ), 7/zM ( ), 2/~M ( ) 597

4 BOCHEMSTRY ond MOLECULAR BOLOGY NTERNATONAL Fluorescence spectrum of NYS and AMP: The effect of HSA s also a revealed on the exctaton spectra of both antbotcs, as shown n Fg. 3. For NYS (Fg. 3-A), the alteraton observed upon ncrease of albumn concentraton s dfferent from the ones observed n the absorpton spectra beng the magntude of change dependent on the albumn concentraton. An ncrease n ntensty at hgher energes s observed (282 nm), wth a red shft of about 1 nm n the presence of albumn. Smlar alteratons are observed on the exctaton spectrum of AMP (Fg. 3-B). An ncrease of the ntensty wth a red shft from 28 nm for the free antbotc to about 3 nm for the bound-proten antbotc s shown. Two lttle peaks at 325 nm and 365 nm were also observed. Emsson of the fluorescence propertes of NYS and AMP are shown n Fg 4 (A-B), n the absence and presence of ncreasng concentratons of HSA. t can be seen that both polyene antbotcs have an emsson of the fluorescence wth a peak at 42 nm for NYS, whle AMP showed two peaks of emsson at 447 nm and 475 nm, wth no shft at ther poston. However, the AMP peaks rato (445 nm/475 nm) dd not reman constant at ncreased concentratons of HSA, showng a varaton from.91 n the absence of proten to 1.4 at saturaton of the albumn-amp bndng stes. Polarzaton of the fluorescenee: Ttraton curves of the polarzaton of the fluorescence (p) of the polyene antbotcs vs. ncreasng concentraton of the HSA was performed, (data o.5 z L.,J C).4 [22 o.3 _.J,.2 ~ ['; '' 4. A 9. / t ll ~ -, "~,,.1 ~:b ; WAVELENGTH (nm) b.j (D, U3 L~ ry o._j h_ ;> F- _J b.j ry.2 O.1 B / /!! / t r!,' ',!; ',. / ~ - 2 :, WAVELENGTH (nm) Fg. 3: Exctaton spectra of NYS(A) and AMP (B) n the presence and absence of HSA. Proten concentraton ( ) none, (-- --) 1/xM, ( ) 2/zM. 598

5 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL.8 1. Lt. r w.6 (o co L3 rv o D.4 _A D_,2 LLJ ' /! / A / 9 /' / # L", q ", ~. r ',,L, b_l.b (9 LtJ C) LO.6 O.d t- L.4 > --. _1 h FF.2 B./"- "% "~ ' e' t ~s -% ' t 9 9 t,'/ -, t,'/ ""~,, ' s" s 9.,,'7 P. aso WAVELENGTH (nm).o r t WAVELENGHT (nm) Fg. 4: Emsson fluorescence spectra of the NYS (A) and AMP (B) n the absence and presence of ncreasng concentratons of HSA. Polyene antbotc concentraton 2/xM, HSA concentratons: none ( ), 1/zM ( ), 2/zM ( ). Sodum phosphate buffer medum 1 mm, ph 7.4 ram. Temperature 2~ not shown). AMP undergo a large ncrease n p values upon changng from the free (p=.6) to the bound-proten antbotc (p=.22), whle NYS dd not show any aprecable change. Bndng ofpolyene antbotcs to lsa: the proten at a constant concentraton (1/xM) was ttrated wth NYS and AMP. Ther concentratons vared between to 2/zM. The enhancement of the ntrnsc fluorescence of the antbotc was measured at 42 nm for NYS and 447 nm for AMP (Fg. not shown). From the values of the fluorescence vs. the total lgand concentraton, the rato: bound lgand to total proten concentraton (r) was calculated as prevously reported (1), then the Scatchard plots were obtaned and shown n Fg. 5 A- B. NYS showed convex Scatchard plots, whch suggests the presence of postve cooperatvty. Ths effect was enhanced at ncreasng temperatures, as t s demonstrated by the dependence of the Hll coeffcent on the temperature (see Table ). AMP yelded concave non-lneal Scatchard curves, suggestng the presence of heterogenety of bndng stes n HSA to bnd ths lgand. Applyng non-lneal fttng, the n and ther K values were calculated as shown n Table, also the bndng was assayed 599

6 BOCHEMSTRYond MOLECULAR BOLOGY NTERNATONAL.5 25 A u lu u u UUnunll~ B o~ v V~ 7 O,O ~ 1 2,3 r r r Fg. 5: Scatchard plots for the bndng of polyene antbotcs to HSA. NYS (A) Temperature (T) 2~ and ( 9 ) 5~ and AMP (B) Temperature (o) 19 ~ and (V) 42 ~ C. Sodum phosphate buffer medum 2 mm, ph 7.4 TABLE 1 Bndng and thermodynamc parameters for the HSA-polyene antbotc studes complex formaton determned at ph 7,4. The values are the means of four ndependent measurements T nj K n~ K 2 AH ~ AS ~ (~ (1 6 M 4) (1 6 M 4) (Kcal/mol) (e.u.) NYS _ _+, # 17 _ _+.7 AMP 42 2,4 _ _ _ _ _+.,56 _+.1 (-3) (-26) 19#.48 _ _+ 7 The values n brackets are for the secondary bndng stes of AMP. u s the cooperatvty coeffcent # measured at ph 4.3 n acetate buffer. wth HSA postvely charged (at ph 4.3). The thermodynamc parameters assocated to the bndng of these lgands were also calculated (Table ). Fatty acds effect on the bndng of NYS and AMP to HSA: t was selected palmtc and lnolene acds as probes to tested the effect of fatty acd (FA) on the bndng of polyene antbotcs to HSA (1). Fg. 6 shows the dependence of the ntrnsc fluorescence of NYS and AMP bound to HSA n absence and presence of ncreasng concentraton of fatty 6

7 BOCHEMSTRYand MOLECULAR BOLOGY NTERNATONAL 2OO bj o 15 (D 1 LL! Q:~ -- 5 u_ Q F-...J ha ~ O 5 4 [FA]/[HSA] 5 Fg. 6: Effect of lnolec (square symbols) and palmtc (crcle symbols) acd on the fluorescence of bound NYS and AMP to HSA. Flled symbols: NYS, hollow symbols: AMP. Polyene antbotcs concentraton 9/~M, HSA concentraton 5/zM, medum sodum phosphate buffer 2 mm, ph 7.4. Temperature 2~ acds. The data have been plotted as percent of fluorescence varaton respect to the fluorescence of bound polyene atbotc n absence of fatty acd. t can be seen that palmtc acd produced a dsplacement of the polyene antbotc bound to albumn at low FA/HSA rato, whle at hgh rato an enhacement of the bndng can be seen. Only lnolec acd produced a contant ncreasng of the bound fracton of antbotcs DSCUSSON Some unsaturated lnear fatty acds have been used as probes to measure conformatonal changes n proten, such as cs parnarc (5, 6) and retnoc acd (1). The macrolde polyene antbotcs flpn and amphotercn B have been also used as optcal probes (8) to detect conformatonal change n membranes and lyposomes wth specal reference to the polyene-cholesterol nteracton. The absorpton spectra of both antbotcs are not dentcal, AMP shows a more complcated absorpton spectrum due to the presence of a major number of unsaturated bounds. Only AMP showed a senstve modfcaton on ts absorpton spectrum. The dpolar nteracton between the transton dpole of the AMP and some amnoacd resdues present at the bndng stes n HSA produce a dsmnuton of the energy of the fluorescence emsson, suggestng a dfferent ground state of the AMP when ths lgand s bound to HSA, whle NYS dd not showed ths effect. The requrements for excton nteracton are very strct, dependng on the 61

8 BOCHEMSTRY ond MOLECULAR BOLOGY NTERNATONAL relatve orentatons and dstances of the transton moments of the partcles, ths revealng the structural dfferences between AMP and NYS. The greater ntenstes of the lower energes components of the exctron mples a parallel arrargement between the chromophore and the local dpolar moment of the albumn bndng stes. The parameters that have been used to evaluate the antbotc-sterol nteracton for AMP are the -~ and 2~ transtons ntensty (2) The rato of the fluorescence ntenstes between 437 nm and 475 nm, whch dd no reman constant n the presence of albumn, suggestng that the second transton s more perturbed than the frst due to the change n the polarzablty n the envronment of the lgand. AMP was very senstve to the rgdty of the medum, because a large ncrease n the polarzaton was observed. Regardless of the compounds studed here, the shape of the Scatchard plots suggest a dfferent bndng mechansm for both lgands. AMP showed two types of bndng stes, the frst havng very hgh affnty, around M-~, and the secondary bndng ste wth an affnty one order lower than the prmary. The ncrease of the temperature reduced the affnty of the secondary bndng stes n a sgnfcant manner, renderng the nteracton of ths ste wth the AMP non-specfc. The convex form of the Sactchard plot for NYS s suggestng the presence of a postve cooperatve process whch s temperature dependent, as s observed by the low value of the Hll coeffcent (y) at low temperature. From the thermodynamc pont of vew some dfferences can be observed between the two lgands. An electrostatc component s observed for NYS and AMP albumn complex formaton judgng by the negatve AH values that both lgands yelded. The AS values observed were largely postve, suggestng a very mportant dsorder that brought about at the proten bndng stes by NYS and AMP bndng, however, the secondary bndng ste for AMP showed negatve AS value, whch suggests a dfferent bndng mechanm for ths ste from that of the frst. The HSA affnty for both antbotcs was sgnfeatvely modfed wth the proten havng postve electrcal charge (ph 4.3), confrm the presence of an electrostatc component n the nteracton. At low faty/proten rato, palmtc acd produced a dsplacement of both antbotcs from ther bndng stes on albumn, whle at hgh rato an ncreasng of the bndng was observed, suggestng that ths fatty acd s bound to the prmary bndng ste of both antbotc. Lnolec acd (a non-saturated fatty acd) produced an ncreasng n the affnty of albumn for both antbotcs, suggestng that ths fatty acd s bound near the bndng stes of NYS and AMP. 62

9 BOCHEMSTRY ond MOLECULAR BOLOGY NTERNATONAL REFERENCES 1. Nerl, Pc6 (1994) Mol. Bol. nt. 32, Castanho, M., Preto, M. and Acuha, U. (1996) Boehm. Bophys. Aeta 1279, Omura, S. Tanaka, H. (1984) Macrolde Antbotcs: Chemstry, Bology and Practce, p 351, Academc Press N.Y. 4. Wyman, J. and Hll, S. (199) Bndng and Lnkage. Unversty Scence Books Ed. Calforna 5. Sklar, L., Hudson, B., Pctersen, M. and Damond, J. (1977) Bochemstry 16, Sklar, L. Hudson, B. Smon, R. (1977) Bochemstry 16, Castanho, M., Contnho, A. and Preto, M. (1992) J. B. Chem. 267, Bttman, R., Chen, W. and Anderson, R. (1974) Bochemstry 13,