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A fluorometric study of the interaction of bradykinin with lipids

A fluorometric study of the interaction of bradykinin with lipids
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  278 Biochimica et Biophysica Acta, 997 (1989) 278-283 Elsevier BBAPRO 33419 A fluorometric study of the interaction of bradykinin with lipids A.G. Appu Rao 1, John M. Stewart 1, Raymond J. Vavrek 1, Laurel O. Sillerud 2, Nancy H. Fink 2 and John R. Cann 1 t Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, CO and z Biomedical NMR Facility, Division of Life Sciences, Los Alamos National Laboratory, University of California, Mail Stop M880, Los Alamos, NM U.S.A.) (Received 22 March 1985) Key words: Bradykinin; Cerebroside sulfate; Phosphatidyi inositol; peptide-lipid interaction; Fluorescence The interaction of bradykinin (BK) with lipids has been followed by steady-state fluorescence measurements. Addition of either cerebroside sulfate (CS) or phosphatidylinositol (PI), solubilized with the nonionic surfactant C12E8, to BK or its analogue [Gly6].BK enhances the relative fluorescence intensity of peptide emission at 288 nm. Fiuorometric titration of the peptide with lipid has been used to quantitate the interactions in terms of stoichiometry and equilibrium constant. Job's method of continuous variation for the BK-CS interaction gave a stoichiometry of 1: 2 for the complex. The value of the equilibrium constant, K, for the interaction of either BK or [Gly61-BK with CS is 1.5.10 4 M-l. The BK-PI interaction is weaker; K-5.0- 103 M-I. Although electrostatic forces no doubt play a major role in these interactions, measurements on the model poptide Giy-Phe-Giy indicate that the phenylalanine residues of BK are disposed in the hydrophobic environment provided by the lipid-C12E8 mixed micelle. 13C-NMR measurements on [99 t3Ce-Gly6]-BK show that there is no change in its e/s/trans ratio upon interaction with CS. The increase in the relative fluorescence intensity of BK accompanying its cooperative interaction with sodium dodecyl sulfate (SDS) implicates the role of hydrophobic forces in this interaction as well. These results bear on the interpretation of the changes in circular dichroism (CD) of BK caused by SDS. Introduction The peptide hormone bradykinin (Argl-Pro2-Pro 3- Gly4-PheS-Ser6-Pro7-PheS-Arg9) has been implicated in many pathologic conditions in man [1]. BK exerts its biological effects by binding to specific receptor sites on the cell membrane, thereby triggering a series of poorly understood biochemical events which are manifested among other things by pain, contraction of smooth muscle, and lowering of arterial pressure [1]. Nor are the structures of the receptor sites known, but it can be inferred, for example, that they probably contain anionic groups which interact electrostaticaIly with the cationic peptide, neither des ArgLBK nor des-Argg-BK possess- hag BK-like activity [2]. Conceivably, acidic lipids may be a component of the receptors [3-5] or interaction of BK with membrane lipids might enhance its binding to Abbreviations: BK, bradykinin; CS, cerebroside sulfate; PI, phosphat- idylinositol; SDS, st .ium dodecyl sulfate; CD, circular dichroism. Correspondence: J.R. Cann, Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, B-121, 4200 E. 9th Avenue, Denver CO 80262, U.S.A. the receptor, as espoused for regulatory peptides by Schwyzer [6,7]. Previously [8], we used circular dichro- ism to demonstrate and partially characterize the inter- action of BK with acidic lipids which bear a net electri- cal charge; e.g., cerebroside sulfate (CS) and phosphat- idylinositol (Pl). In contrast, it was found that BK does not interact with lipids bearing no net charge; e.g., cerebroside and phosphatidylcholine. These studies have now been extended to fluorometric determination of the equilibrium association constant for BK-CS and BK-PI interactions. As before, the interactions are with mixed micelles of the lipid and the nonionic surfactant used for its solubilization. The highly ordered micelles, whose diameters [8] are predominantly 400-600 A,, have the advantage over vesicles in being thermodynamically sta- ble [9]. Also, in mimicking cell membranes they present surface anionic groups for electrostatic interaction with the cationic groups of BK and provide a hydrophobic environment for interaction with the aromatic group of the Phe residues of the peptide. Materials and Methods BK, its analogues [GIy6]-BK and [99% 13Ca-Gly6]-BK were synthesized by the solid phase method, purified 0167-4838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)  and characterized as described [10]. CS was obtained from Supelco, Inc., Bellefonte, PA, and PI sodium salt from Sigma Chemical Co, St. Louis, MO. The purity of CS was confirmed as reported previously [8]. Sodium dodecyl sulfate (SDS) Electrophoresis Purity reagent was from Bio-Rad Laboratories, Richmond, CA, and the nonionic surfactant oeta-ethyleneglycol mono- n-dodecylether (C12E8) was from Nikko Chemical Co, Ltd. Tokyo, Japan. Glycyl-L-phenylalanylglycine mono- hydrate was a product of Cydo Chemical, Los Angeles, CA. Peptide solutions were prepared from weighed lyophilized quantities; concentratio~s were based on analysis for peptide content. CS and PI were solubilized with C12E8, following the method of Shirahama and Yang [11]. The weighed lipid and C12E8 were dissolved in chloroform/methanol mixture (2:1 (v/v)) and ro- tary evaporated to dryness. The flash dried material was dissolved in glass-distilled water, followed by a low power (20 mW) brief (20 s) sonication at room temper- ature under dry nitrogen atmosphere. The resulting mixed micelles were characterized previously [8] with respect to size and structure by electron microscopy. Fluorescence measurements were made on a Farrand Mark I spectrofluorometer. The temperature of the cell was maintained at 27 + 0.02 ° C, utilizing a circulating water bath. Fluorescence emission spectra of 3.8- 10-5 M BK in the absence and presence of graded concentra- tions of lipid were recorded using an excitation wave- length of 266 nm. Excitation and emission slits were 4 and 8 nm, respectively. The enhancement of the fluor- escence intensity of BK by lipids was corrected em- pirically for internal absorption by subtraction of the fluorescence intensity shown by the same concentration of lipid in the absence of BK. The enhancement of relative fluorescence intensity at 288 nm by lipids was analyzed in terms of binding of lipids by BK, using established procedure [12]. Under the assumptions that the binding of each lipid molecule produces the same degree of fluorescence enhancement and that the bind- ing is statistical, the intrinsic binding constant, K, is given by the equation K = fl 1 (1) (1-#) G in which fl= Q//'Qrnax and Cf= C-hilT, where Q is the corrected fluorescence intensity; Qmax, the maximal fh,,~rescence enhancement; Cf, the molar equilibrium concentration of unbound lipid; C, the molar con- stituent concentration of lipid; n, the binding stoi- chiometry; and T, the molar constituent concentration of BK. The value of K is given by the slope of the plot of fl/ 1- fl), versus Cr. Qmax was determined by ex- trapolation of a double-reciprocal plot of Q versus C to the intercept. The best fit to the data was obtained by 279 the method of least squares on a computer. 'The value of n for the BK-CS interaction was obtained by Job's method of continuous variation [8,13]. The total con- centration of BK plus CS was held constant at 4.0.10-4 M and their relative proportions varied. The percentage change in fluorescence intensity was plotted against the mole fraction of CS. These data were computer-fitted to a 5th degree polynomial, and the maximum was de- termined numerically in the usual manner. The re- sultant stoichiometry of the BK-CS complex for- tuitously agreed with the value obtained by the method of 'limiting slopes'. The former analysis of the data is displayed under Results and Discussion because it is rigorous; the frequently used method of 'limiting slopes' has been shown not to be theoretically justifiable [14]. Fluorescence polarization measurements were made with a SLM-Aminco Spectrofluorometer Model 48000 TM fitted with a water-cooled photomultiplier tube housing, the temperature being maintained at 27 _+0.02°C, utilizing a circulating water bath. The data were ob- tained by setting the excitation and emission wave- lengths at 266 and 288 nm, and were analyzed on a computer. Both the excitation and emission slits were 8 nm. Two concentrations of BK (3.8.10-5 M and 7.6- 10 -5 M) gave virtually the same results and accordingly were averaged. The intensities of horizontal and vertical components of the emitted light (Ill and I L) were corrected for the contribution of scattered light de- termined with a referenced CS concentration in the absence of BK. The grating correction factor, G, that corrects for wavelength-dependent distortions of the polarizing system was obtained using G ( FHv ] and Ill ~, F.. 1 ~ ( P,v] where Fvv, Fvn, Fnv, Full are the fluorescence inten- sity components, in which the subscripts refer to the horizontal (H) or wertical (V) positions of the excitation and emission polarizers separately. The anisotropy was calculated using the equation A-- I )_1 11 ,, ix )+2 (2) Proton-decoupled ~3C-NMR spectra were obtained on a Bruker AM400wb spectrometer operating at 100.61 MHz in the Fourier transform mode. A 22727 Hz sweep was accumulated in 16384 data points resulting in a digital resolution of 0.014 ppm/pt and an acquisition time of 0.36 s. A 38 ° (6 #s) pulse width was followed with a relaxation delay of 1.0 s; the total number of scans for each spectrum was 22 000. Any nuclear Over- hauser effect was suppressed by decoupling with 2 W  Results only during acquisition. The samples were spun at 9 Hz and thermostated at 301 K. Chemical shifts were refer- enced to external 0.1~ (v/v) p-dioxane in D20 at 67.4 ppm downfield from TMS. The spectra were processed with a 5 Hz line broadening prior to Fourier transfor- marion and the peaks were fitted with the aid of NMR1 software using the sum of Lorentzians (New Methods Research, Inc.). Spectra were obtained on 0.7 mM peptide in H20 solutions containing either 2.0 mM C12E8 or 2.0 mM C12E8/0.7 mM CS. B The interaction of BK and its analog [GIy6]-BK with lipids was monitored by following the change in relative fluorescence intensity of the Phe residues pro- 50 40 30 20 10 O= 280 C D 60 c b 50 4O 30 2O 10 0 , I I I I I J I , I 280 300 320 280 300 320 WAVELENGTH (nm) Fig. 1. Effect of lipids on the fluorescence emission spectrum of BK and [GIy6]-BK, and of solvent on the emission of the model peptide Gly-Phe-Gly. (A) Interaction of 3.81.10-5 M BK with the following concentrations of CS: curve a, 0.0; curve b, 8.26.10 -5 M; curve c, 3.3 10 -4 M. (B) Interaction of 3.49.10 -5 M [GIy6]-BK with CS: curve a, 0.0 lipid; curve b, 9.9.10 -5 M; curve c, 1.98.10 -4 M. (C) Interaction of 6.62.10 -5 M BK with PI: curve a, 0.0 lipid; curve b, 1.10 -4 M; curve c, 2-10 -4 M. (D) Spectrum of 1.5.10 -4 M Gly- Phe-Gly in water (curve a) and in methanol (curve b); the same result was obtained with 3.0-10 -4 M peptide, in conformity with Beer's law. Molar ratio of lipid/C12E8 = 3.7 in panel (A) and (B), 3.4 in panel (C); pH 4.7-4.9. INTERACTION OF BK WITH CS 50~ O. 12r~~ o o ;/ + oF ../ I °°T / '°oot / 2°1" 7 I ° I/ °°TF o 0 ~ 0.00~ 0 0,1 0.2 0.3 0.4 05 0 1 2 3 4 C, CS (mM) 10 -4 x I/Ccs 20 I0 8 + : : 'p ./__. . 0 0.1 0.2 0.3 0.4 0.5 Mole Fraction of CS Ct, (mM) Fig. 2. Quantitation of the interaction of BK with CS. A) Percentage enhancement of fluorescence intensity, Q, as a function of constituent lipid concentration, Ccs; T= 3-81 10-5 M. (B) Double-reciprocal plot of data in panel (A); Qmax = 47 + 3.1 (+ indicates probable error in all cases). (C) Job's plot, CBK +Ccs = 4.0 10 -4 M. (D) Mass action plot of data (panel A) in accordance with Eqn. 1. duced by graded concentration of lipid. The salient features of the resulting emission spectra, Fig. 1A, B and C, are (a) the lipid-induced enhancement of fluor- esence without detectable change in wavelength, 288 nm, of maximum emission and (b) insignificant changes in band shape. Control experiments established that C12E8, the nonionic surfactant used to solubilize the lipid, had no effect on the fluorescence emission of the peptide over the concentration range used in these measurements. Quantitation of the BK-CS interaction is displayed in Fig. 2. As shown in Fig. 2A, 0.5 mM CS enha~.ced the fluorescence by 4e%, which corresponded tc~ 85~ completion of the reaction as deduced from the linear double-reciprocal plot of Q vs. C'cs, Fig. 2B. The stoi- chiometry of the BK-CS complex was estimated from the Job's plot, Fig. 2C, to be 1 : 1.7 + 0.2. U~ing a value of n = 2 and extent of reaction reckoned from Fig. 2B, the mass action plot presented in Fig. 2D was con- structed in accordance with Eqn. 1. The equilibrium association constant (_+probable error) given by the slope of this plot is 1.5(_+0.12). 10 4 M -]. The value 1.6 _+ (0.13). 10 4 M -], of the equilibrium constant for  281 INTERACTION OF [Gly6]-BK WITH CS • ,o °°T I ' 3 o 1 ~ 3 4 10 4 l~l/C, cs cf (raMs INTERACTION OF BK WITH P, 0.30. / 3.0 f / 0.25 / 2.5 oo. o. / r / o o,0 ,j "°r ,2". 0.050 d'i I I 05 ~l~J~l~L~l~o 0 0.2 0.4 0.6 08 0 1 2 3 0 01 02 0.3 0.4 0.5 06 CPo (raM) 10 -4 x llCp: Cf (mM) Fig. 3. Quantitation of the interaction of [Glyt]-BK with CS and BK with PI. (A) Percentage enhancement of fluorescence intensity, Q, as a function of constituent lipid concentration, C~; T= 3.49.10 -s M for [Glyt]-BK and 6.62-10 -s M for BK. (B) Double-reciprocal plot of data in panel A, Qmax = 64+3.2 for [Glyt]-BK and 42+8.6 for BK; the curve through the BK-PI data points is the least-squares quadratic fit. (C) Mass action plot of data (panel A) in accordance with Eqn. 1. the interaction of the BK analog [Glyt]-BK with CS, Fig. 3 (upper panel under the assumption that n = 2), was the same within experimental error as for the BK-CS interaction. Mention should be made of the fact that Qma,, deduced from the linear double-recipro- cal plot, Fig. 3B upper panel, is 36% larger than in the case of BK. The interaction of BK with PI, Fig. 3 lower panel, differs in two respects from the BK-CS system: (a) for whatever reason, the double-reciprocal plot of Q vs. C-el, Fig. 3B lower panel, is nonlinear and (b) the value, 4.8 +_ (0.39)- 10 3 M-1, for the association constant, Fig. 3C (lower panel under the assumption n = 2), is signifi- cantly lower than for BK-CS. In order to gain insight into the molecular mecha- nism of enhancement of peptide fluorescence by lipids, the emission spectrum of the tripeptide Gly-Phe-Gly was recorded in water and in methanol, Fig. 1D. The less polar methanol enhanced the emission intensity of the Phe residues in this model peptide without shifting the maximum wavelength, 288 nm, and without affect- bag the band shape. Fluorescence polarization measurements were made for the BK-CS system. As shown in Fig. 4, the ani- sotropy of BK was not significantly changed by CS over the concentration range indicated, which means that the rotation of the aromatic ring of the Phe residue(s) of the peptide in the complex is not restricted with respect to unbound BK. 0.04 . el O 0.02 rr. i- 0 O 0.02 < 0.04 0.0 0.1 0.2 0.3 0.4 ~CS (mM) Fig. 4. Plot of fluorescence anisotropy of the BK-CS_ system vs. the constituent concentration of lipid, Ccs.  282 INTERACTION OF BK WITH SDS 280 300 320 WAVELENGTH nm) P 0 O0 80 60 40 20 0 0 r B o 2 4 6 C. sl)s raM) Fig. 5. Interaction of BK with SDS at pH 8.4 as monitored by fluorometry. A) Effect of SDS on the emission spectrum of 3.81.10- 5 M BK: curve a, 0.0 SDS; curve b, 2.10- s M; curve c, 4-10- 3 M. B) Plot of percentage enhancement of fluorescence intensity, Q, vs. constituent concentration of SDS, CSDS- The 13C-NMR of [99g 13C~-Gly6]-BK confirmed [15,16] the unusually high cis/trans ratio about the Gly6-Pro peptide bond of this BK analog. Thus, in the absence of lipid, the chemical-shift difference between the cis and trans resonances of the methylene carbon of Gly 6 was 8 cis-trans)--1.06 ppm and the ratio cis/trans---0.55 __.0.17. No significant difference was observed in the presence of CS; i.e., 8 cis-trans)- - 1.09 ppm and the ratio cis/trans = 0.67 + 0.07. To round out our previous CD studies [8] on the interaction of BK with the model amphiphile SDS, this interaction was also characterized fluorometricaUy. As in the case of lipids, interaction with SDS was mani- fested by fluorescence enhancement without change in wavelength of maximum emission or in shape of the band, Fig. 5A. Consistent with the earlier CD results, titration of BK with SDS as monitored fluorometrically, Fig. 5B, is indicative of a cooperative interaction. Con- struction of a Hill plot, however, is precluded by the previously noted [8] SDS-induced self-association of BK; i.e., the changes in spectroscopic properties of the peptide are most likely not proportional to the amount of SDS bound. Note that the concentrations of SDS in these experiments were below its critical miceUar con- centration (8.2 mM), which is unaffected by BK. Thus, the interaction of BK is with monomeric SDS. Discussion The major feature of this study is the determination of equilibrium association constants for two BK-lipid systems, the values for which are comparable to those for other biologically active peptides [6,17,18]. More- over, fluorometry had the distinct advantage over the earlier CD measurements [8] in that it distinguished between the BK-CS and BK-PI interactions. CD could not distinguish between the two because the change in CD accompanying these interactions is most likely the net result of changes in at least two chromophores, the Pile residues of BK and the lipid. In contrast, the fluorescence measurements essentially reflect only the environment of the Phe residue(s) of BK. The interaction of [GIy6]-BK with CS was examined to see whether the unusually high cis Pro ~ coatent, 35~, of this BK analogue might affect the inv~raction. From the value of the equilibrium constant it is clear that the cis Pro ~ content of the peptide does not influence this aspect of the interaction. Furthermore, the 13C-NMR measurements indicated no significant change in the cis/trans ratio due to the interaction. It is not surpris- ing, however, that there is a difference in the values of Qmax for the BK-CS and [Gly6]-BK-CS interactions, since one might surmise a difference in the geometry of their respective complexes. It was proposed previously [8] that electrostatic inter- actions between the two Arg residues of BK and the anionic groups of the acidic lipids on the surface of the mixed lipid-C12E8 miceUes are important for the bind- ing reaction, but that other forces possibly, hydrophobic in nature, must also be involved. In support of the latter contention the changes in fluorescence emission spectra occasioned by the neuropeptide-lipid interaction (Figs. 1A-C) find qualitative parallel in the fluorescence changes effected the environment of the model peptide Gly-Phe-Gly from water to methanol (Fig. 1D). The conclusion is therefore reached that complex formation results in localization of the Phe residues of BK either on hydrophobic regions of the micellar surface or within the hydrophobic interior of the mixed micelles. This conclusion is not necessarily inconsistent with the ob- servation, Fig. 4, that the fluorescence anisotropy of BK is not affected by interaction with CS. Consider the insertion of the coat protein of bacteriophage M13 into phospholipid vesicles [19,20]. The Tyr residues, which are in the hydrophobic region of the protein, are buried in the vesicle, while the Phe residues in the hydrophilic regions are exposed on the outer surface. Nevertheless, above 24 °C the buried tyrosines are moving as rapidly as the exposed phenylalanine residues. Finally, the cooperative nature of the interaction of BK with the model amphiphile SDS as revealed by earlier CD measurements [8] and interpreted in terms of ligand-mediated self-association of the peptide is con- firmed by the fluorescence data presented in Fig. 5B. Here too, the enhancement of fluorescence cat', d by nonmicellar concentrations of SDS is attributable to hydrophobic interaction of the aromatic ring of the Phe residues(s) with the aliphatic moiety of SDS. Compare Fig. 5 with Fig. 1D. It is apparent from these results that interactive perturbation of the Phe chromophore makes a major contribution (approx. 70~ as judged
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