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The binding of bilirubin to albumin a study using spin-labelled bilirubin
Biochimica et Biophysica Acta, 742 (1983) 341-351 Elsevier Biomedical Press
341
BBA 31444
THE BINDING OF BILIRUBIN TO ALBUMIN
A STUDY USING SPIN-LABELLED BILIRUBIN M A R I O B K A N C A a A L D O G A M B A a.,, PAOLO M A N I T T O b, D I E G O M O N T I c and G I O V A N N A S P E R A N Z A b
a Istituto di Chimica Fisica, Universit,~ di Sassari, V. Vienna 2, 07100 Sassari, b lstituto di Chimica Organica, Universiti~ di Milano, and ¢ Centro per 1o Studio delle Sostanze Organiche Naturali del CNR, Milano (Italy) (Received September 22nd, 1982)
Key words: Spin label," Bilirubin; Protein binding," Human serum albumin; Cotton effect
Binding between human serum albumin and a spin-labelled derivative of bilirubin was investigated by circular dichroism, fluorescence quenching, electron spin resonance and visible spectroscopy. The orders of magnitude of the binding constants obtained by flurorescence quenching and electron spin resonance spectroscopies were 10 7 and 10 a l - m o l - 1, respectively. These data suggest that most spin-labelled bilirubin interacts with human serum albumin at the side not holding the spin-labelled side-arm. CD measurements showed the presence of at least two sites, associated with opposite Cotton effects. It is worthy of note that the Cotton sign of the first site is inverted with respect to the corresponding one of bilirubin. CD measurements on mixed systems (spin-labelled bilirubin/human serum albumin/bilirubin) were also performed. The decomposition of the ternary curves shows that the rotatory power of bilirubin bound to human serum albumin is higher in the ternary system than in the binary (bilirubin/human serum albumin). The corresponding CD measurements for the binding between spin-labelled bilirubin and bovine serum albumin are also reported and discussed.
Introduction
Interactions of bilirubin, the end product of heme catabolism with serum albumin, the most abundant protein in mammalian plasma, have been extensively studied for both theoretical interest and clinical importance [1-5]. Our interest in bile pigment metabolism led us to study the interaction between bilirubin and proteins using suitably synthesized spin-labelled bilirubins. To our knowledge no investigation of such a kind has been reported in the literature, although a great deal of work of different origin has been carried out by means of physical, spec* To whom correspondence should be addressed. 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
troscopic and chemical methods [6,7]. The only spin-labelled molecule reported to be bound to human serum albumin at the highest affinity site for bilirubin is 1-N-(2,2,6,6-tetramethyl-l-oxyl-4piperidinyl)-5-N-(1-aspartate)-2,4-dinitrobenzene [8], i.e., a completely different ligand from bilirubin. Like bilirubin the compound appears to be a suitable clinical tool for determining the concentration of vacant bilirubin binding sites of albumin in serum [9]. We present here the first synthesis of a spinlabelled bilirubin (6) and the specific binding of it to human serum albumin. The structure of spinlabelled bilirubin was planned so that one of the two outer pyrromethenone moieties would have a spin label, thus allowing the other one (and most
342 of the tetrapyrrolic skeleton) to interact with the protein, as is the case with bilirubin binding. Materials and Methods
Bilirubin. Bilirubin (Merck) was purified as previously described [6], and checked for purity by TLC [10] and HPLC [11], giving c = 60700 at 454 nm [6] in CHC13. 4-Amino-2,2,6,6-tetramethylpiperidin- 1-yloxy (3) was obtained from Aldrich. Human serum albumin. Essentially fatty acidfree human serum albumin (Sigma No. A-1887) was used in all experiments. Molar protein concentrations were based on weighing and on a molecular weight of 69000 [1] ( c = 3 6 5 0 0 at 278-279 nm, 25°C, pH 7.4) [12]. Deionized water was redistilled. A 0.1 M phosphate buffer, pH 7.4, was prepared according to Clark and Lubs [13] and used to obtain stock albumin solutions (10 mg/ml, approx. 1.5 • 10 -4 M). These were kept at 0-5°C for a period of time not exceeding 1 week (no significant change of c was observed). Bovine serum albumin. Bovine serum albumin from Merck (Art. 12018) was used, without further purification, as described above for human serum albumin (bovine serum albumin, ~ = 47 000 at 278-279 nm, 25°C, pH 7.4) [12]. Preparation and measurements of solutions. Stock solutions of bilirubin and spin-labelled bilirubin was prepared and used immediately. About 2.5 mg of the pigment were dissolved in 0.25 ml of 0.02 M aqueous NaOH, diluted to 25 ml with distilled water (1.5 • 10 - 4 M ) . The solutions were protected form light during preparation. Exact solute concentrations were determined spectrophotometrically in the presence of an almost equimolecular quantity of human serum albumin at pH 7.4 in 0.1 M phosphate buffer (bilirubin: c = 4 6 5 0 0 at 458 nm; spin-labelled bilirubin: c = 40 800 at 418 nm). Unless stated otherwise, a measured amount of the bilirubin stock solutions was added to a protein solution with stirring, followed by dilution with phosphate buffer (0.1 M) or distilled water, in order to obtain the concentrations and molar ratios between the ligand and the protein desired. Usually the pH of the final solutions was within the range of 7-8. It was adjusted to the value of 7.4 by addition of a few drops of 1 M NaOH or KH2PO 4.
Solutions were protected from light until measurement. Synthesis of spin-labelled bilirubin (_6). 1,3-Propandithiol (0.25 ml) was added to a solution of bilirubin (100 mg) in CHCI 3 (100 ml). After the addition of a few crystals of p-toluensulphonic acid the reaction mixture was stored overnight at room temperature in the dark. Evaporation of the solvent under reduced pressure to 10 ml, followed by addition of MeOH, led to a precipitate which was filtered, washed twice with MeOH and dried. The solid (77 mg) was shown to be pure (2) by TLC (moving more slowly than bilirubin on silica gel plates, benzene/CHC13/MeOH(53 : 45 : 2)) and elemental analysis (calc. for C 3 6 H 4 4 N 4 0 6 S 2 : C, 62.42; H, 6.35; N, 8.00; found: C, 62.34; H, 6.32; N, 7.89). nH-NMR (C2HC13; TMS, 8: 1; 53 d (3H, J = 8 Hz, Me-CH(S-)-C(18)); 1.97s (3H, Me-C(2)); 2, 15s (9H, 3Me); 2.5-3.1m (propionic acid and C(18') side chains); 3.96q (1H, J = 8Hz; Me-CH-(S-)-C(18)); 4.06s (2H, central CH2-); 5.4-5.7rn (CH 2 = CH-); 6.11s and 6.19s (methine bridges); 6.4-6.8m (CH 2 = CH-); 9.26s (2H, pyrole NHs); 10.69s and 10.77s (2H, amide NHs). N-Iodoacetoxylsuccinimide (_4) was prepared according to the procedure of Hampton et al. [14] in 60% yield, m.p. 148°C (literature [14], 148-150°C; calc. for CrHrlN404: C, 25.44; H, 2.14; N, 4.95; found: C, 26.55; H, 2.36; N, 5.20). nH-NMR (DMSO-d 6, TMS, 8): 2.81s (4H); 4.1s (2H). N-(2,2,6,6-Tetramethyl-4-piperidin- 1yloxy)iodoacetamide (_5) was obtained by adding compound (4) (1 g dissolved in 20 ml H4furan and 3 ml phosphate buffer [13], pH 7.5) to a solution of 4-amino-2,2,6,6-tetramethylpiperidin- 1-yloxy (3) (500 mg) in 10 ml H j u r a n . After 30 min stirring at room temperature in N 2 atmosphere, the reaction mixture was extracted with CHCI 3 and the organic phase washed with dilute HCI, and dried on anhydrous sodium sulphate. Evaporation of the solvent under reduced pressure gave a red oil, which was chromatographed on silica gel column eluting with 1% MeOH in CHCI 3. Fractions containing pure (5), as shown by TLC (silica gel plates, C H C l J E t O H 5 : 1), were evaporated and the residue crystallized from benzene, affording dark orange needles (m.p. 113-116°C, lit. 114-117°C [15]); (calc. for C 1nH20IN202: C, 38.95; H, 5.94; N, 8.26; found: C, 39.0; H, 5.8; N, 7.9).
343 To obtain the spin-labelled bilirubin (6) the SHbearing bilirubin (_2) (30 mg in a mixture of 10 ml N, N'-dimethylformamide and 1.5 ml 0.1 M phosphate buffer, pH 7.8) was treated with compound (5) (20 mg in 0.5 ml dimethylformamide). After 1 h at room temperature under a slow stream of argon the reaction mixture was poured into water (20 ml) and extracted with CHC13 (25 ml) after the addition of an excess of solid carbon dioxide. The organic phase was then dried on anhydrous sodium sulphate and evaporated to a small volume (approx. 0.5 ml) in a vacuum. The precipitate resulting from the addition of methanol was collected (25 mg) and shown to be spin-labelled bilirubin (6) by 1H-NMR (C2HC13, TMS, 8): 1.53 d (3H, J = 8 Hz, Me-CH(S-)-C(18)); 1.99s (3H,MeC(2); 2.16s (3Me); 4.05s (central bridge largely hiding the quartet due to Me-CH-(S-)-C(18)); 5.4-5.7m (CHE=CH-); 6.14s and 6.21s (two singlets due to the methine bridges); 6.4-6.8m (CH 2 = CH-); 9.285s, (pyrrole NHs); 10s and 10.80s (amide NHs); all the protons C-H present in the C(18') side-chain gave rise to very broad peaks buried under the sharp signals of the biladiene substituents in the range of 1.3-3.3. The purity of (6) used in spectroscopic measurements was checked by TLC (benzene/CHC13/MeOH (45 : 45 : 10)) and elemental analysis. Its ESR spectrum in phosphate buffer, pH 7.4, is displayed in Fig. 2(a). Elemental analysis calc. for C 4 7 H 6 3 N 6 0 8 S 2 : C , 62.43; H, 7.02; N, 9.29; found: C, 62.11; H, 6.88; N, 9.15. ESR. The experimental samples (70 #l) were prepared by combining stock solutions of 5. 10 -4 M human serum albumin (0.1 M phosphate buffer, pH 7.1), and 1 • l 0 - 2 M spin-labelled bilirubin (0.5 M NaOH) with a micropipette (50 and 10 #l). The final pH values of the solutions were 7.4-7.5; after mixing the samples were transferred by capillarity into a disposable pipette (Coming) which was used for ESR measurements. The concentration of the spin-labelled bilirubin, the spin-labelled arm of which is free in solution, was proportional to the height of the high-field peak of the sharp triplet. Since the total amount of spin-labelled bilirubin was known, the amount of spin-labelled bilirubin bound to serum albumin could be determined. Absorption spectroscopy. The experimental sam-
pies held about 1 • 10-5 M concentration of spinlabelled bilirubin, or bilirubin and variable concentrations of human serum albumin. The spectra were recorded in the range 350-550 nm with a Varian 634 spectrophotometer. Fluorescence quenching. The measurements were performed with an Aminco-Bowman spectrofluorimeter. The work-exciting wavelength was 294 nm and that of emission 340 nm. Three cuvettes were used in the experiment. One, containing phosphate buffer (0.1 M, pH 7.4), was used as zero percent reference; the second one, containing 2.5 • 10 -6 M human serum albumin was used as 100% fluorescence, and the third one holding exactly 2 ml human serum albumin was that used for the titrating solution. A solution of spin-labelled bilirubin (or bilirubin), at a concentration 20-times greater than that of the human serum albumin was added to the third cuvette. Then the fluorescence of the three solutions was measured and the procedure was repeated at least twenty times after adding 10 #1 of the spin-labelled bilirubin (or bilirubin) solution. All the measurements were corrected for the inner filter effect, and for dilution. Binding equilibria were analyzed by the method of Levine [16]. Circular dichroism. The experimental samples held 2.5.10-SM concentrations of serum albumin and variable concentrations of spin-labelled bilirubin and bilirubin. The spectra were recorded in the 350-700 nm range with a Dicrograph Mark III Jobin-Yvon. Cells of 1 or 0.1 cm path-length were employed. Results and Discussion'
Spin-labelled bilirubin (6) was obtained in satisfactory yields according to the convergent synthesis shown in Scheme I. The functionalization of the spin-labelled bilirubin side-chain at the 18 position was achieved taking advantage of the known acid-catalyzed regiospecific addition of nucleophils to an exo-vinyl group [10]. The structure of spin-labelled bilirubin was affirmed by its NMR spectrum, which also indicated strong conformational analogies between spin-labelled bilirubin and bilirubin in solution. Thus, the spinbearing side-arm in spin labelled bilirubin appears to have only slight effect on the conformational
344
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Fig. 1. Klotz's double reciprocal binding plot for titration of human serum albumin (2.10 .6 tool-1 -I) with spin-labelled bilirubin(e) or bilimbin(~.) by fluorescencequenching. ? is the number of moles of spin-labelled bilirubin or bihrubin bound/mole human serum albumin. Cr is the number of moles of spin-labelledbilirubin or bilirubin which are free in solution.
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stabilization of the a , c - b i l a d i e n e skeleton, which c a n a s s u m e the ridge-tile form typical of bilirubin-like c o m p o u n d s [17,18].
K 2 = 1.3- 106 1. m o l - ] , n 2 = 1.62 for spin-labelled bilirubin, a n d K 2 = 6.7- 106 1 • mol - I , n 2 = 1.19 for bilirubin, were o b t a i n e d p e r f o r m i n g a titration at a h u m a n serum a l b u m i n c o n c e n t r a t i o n higher t h a n ( 1 - 1 . 5 ) . 10 -5 M. U n d e r these c o n d i t i o n s es-
Fluorescence quenching The technique employed, which allows det e r m i n a t i o n of both b i n d i n g affinity a n d b i n d i n g capacity, is described in the Materials a n d Methods section. Even though the b i n d i n g constants o b t a i n e d from fluorescence q u e n c h i n g for the complexes of b i l i r u b i n with h u m a n serum a l b u m i n are easily available in the literature [19], it has been explicitly reported that the reproducibility of the m e a s u r e m e n t s was poor [16]. So, within the scope of a significant c o m p a r i s o n between the b i n d i n g of spin-labelled b i l i r u b i n a n d b i l i r u b i n u n d e r c o m p a r a b l e conditions, the fluorescence q u e n c h i n g for the b i l i r u b i n / h u m a n serum alb u m i n system was re-measured. The reciprocal b i n d i n g plots of Fig. 1 show the existence of two sites at least. According to Klotz a n d H u n s t e n [20], the following parameters were o b t a i n e d : K I = 2 . 8 7 . 1 0 7 1 • mo1-1, n 1 = 1, for spin-labelled bilirubin; K l = 1.26.107 1 • m o l - l, nl = 0.94, for bilirubin. The parameters relating to the second site,
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Fig. 2. ESR spectra for (a) spin-labelled bilirubin in 0.1 M phosphate buffer at pH 7.4; (b) human serum albumin added to solution (a) at r = 0.4; (c) solution (a) frozen at - 18°C; (d) frozen solution (-18°C) of spin-labelled bilirubin in 0.1 M phosphate buffer at pH 7.4; r = 2.85; r = spin-labelled bilirubin/human serum albumin.
345
sentially no unbound bilirubin is detectable until the first site is saturated [16]. Within the limits of the type of interaction detectable by the present technique, it appears that the bindings of spinlabelled bilirubin and bilirubin to human serum albumin are comparable. However, on the basis of the data obtained from the fluorescence measurements reported in Fig. 1, we cannot assert that spin-labelled bilirubin and bilirubin occupy the same site of high affinity of human serum albumin, a n d / o r in the same manner. By considering that the interaction is very similar one cannot exclude the possibility either that the two sites for bilirubin and spin-labelled bilirubin are very close (i.e., at a comparable distance from tryptophan), or that the same site is differently engaged. Electron spin resonance
A representative spectrum of a solution of a spin-labelled bilirubin in phosphate buffer at pH 7.4 is shown in Fig. 2(a). The hyperfine pattern is typical of a nitroxide spin label, and a moderate anisotropy of the hyperfine components is evident in the spectrum. Simulation through a least-square
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fitting [21] gives the following spectral parameters: the nitrogen hyperfine splitting a N(N°)= 17.18 + 0.04 G; the linewidth W = 1.67 + 0.08 G, and a value of 0.13 G for B(N), a linewidth parameter [22] describing the mobility of the spin label. Simulation of the spectrum of a sample of the same free spin label gives the following spectral parameters: a N(NO) = 17.90 + 0.02 G, W = 1.42 + 0.05 G, B(N) = 0.045 + 0.027 G. It is interesting to observe that the mobility in the same solvent is reduced by about three times when the label is bound to bilirubin. The ESR spectrum measured in the same solution in the presence of human serum albumin (r = spin labelled bilirubin/human serum albumin = 0.4) is shown in Fig. 2(b). The spectrum is qualitatively different, since two hyperfine splitting patterns, one typical of a bound fraction and the other associated with a more mobile species, can be observed. The magnitude of the splitting of the nitrogen belonging to the bound fraction is 2T~z = 69 G. A rought measurement of correlation time "r, according to a technique proposed by Freed [23], gives "r --- 2 . 1 0 -8 s. It appears that the spin-labelled chain is not completely immobilized. The hyperfine splitting constant of the nitrogen of the weakly bound fraction is identical (a N(N°) = 17,18 G) to the corresponding one of spin-labelled bilirubin in phosphate buffer solution in the absence of human serum albumin. The coincidence is significant and proves that the spin label either belongs to a fraction not implied in the binding with human serum albumin, or is in the oxydipyrromethene moiety of spin-labelled bilirubin interacting with human serum albumin faced to the water phase. As a matter of fact it is well known that the magnitude of the hyperfine splitting constant of the nitrogen of the nitroxide is strongly dependent on the dielectric constant and on other physical properties of the environment (see for example Ref. 24). The ESR spectrum of spin-labelled bilirubin in the glass phase recorded at - 1 8 ° C in phosphate buffer is shown in Fig. 2(c). The spectrum measured under the same conditions after the addition of human serum albumin to the solution (r = 2.85) is reported in Fig. 2(d). The upper spectrum (c) in Fig. 2 is broadened by spin-spin interactions, which depend on spin concentration, probably due to the
346
formation in solution of micelles of spin-labelled bilirubin [16]. The addition of human serum albumin probably leads to the breaking of its colloidal structure, so that spin-spin interactions responsible for the broadening are greatly reduced. The amount of spin label, free plus weakly bound to the macromolecule, was obtained through the calibration of the high-field peak of the sharp triplet [9]. The amount of strongly bound component was obtained by difference. According to the technique proposed by Wood and Hsia [9], the double reciprocal binding plot described by Klotz and Hunston [20] was built up using a series of ESR spectra measured at different values of r. The experimental points and the plot are shown in Fig. 3. The derived binding constant, which involves only the spin-label moiety, is 6.6-10 3 l" tool -1, the number of sites being n = 1. It is noteworthy that the presence of bilirubin (bilirubin/spin-labelled bilirubin/human serum albumin = 1 : 1 : 1) does not modify the magnitude of the binding constant of spin-labelled bilirubin to human serum albumin. This binding is lower by about four orders of magnitude that that derived from fluorescence quenching (see also Ref. 25). To correlate the results obtained by fluorescence and ESR measurements it has to be considered that the interaction is governed by an equilibrium process (Kn,or = 2.87" 1 0 7 1 • mol -I, K E S R ---- 6.6 • 1 0 3 1 • m o l - l) and that significantly different amounts of spin-labelled bilirubin and human serum albumin (fluor: 10 -6 M; ESR: 10 -4 M) were necessary to obtain the best answers by the two spectroscopic techniques. The observed difference of the K equilibrium constants has to be ascribed to the different quality of bound component detectable by the two spectroscopic techniques. ESR detects only the part of spin-labelled bilirubin whose spin-labelled arm is blocked, but cannot distinguish between spin-labelled bilirubin interacting with human serum albumin through the unsubstituted side and the free spin-labelled bilirubin. Fluorescence detects the total bound component. For example, at concentrations of human serum albumin = spinlabelled bilirubin = 1 0 - 6 mol- 1 - 1, it emerges from Knuor that 80% of spin-labelled bilirubin is bound, whereas from K E S R only 0.5% of spin-labelled bilirubin appears bound. In view of this it is easy
to deduce that most of the spin-labelled bilirubin interacts with human serum albumin through the side without the spin-labelled chain.
Visible absorption The electronic spectrum of spin-labelled bilirubin in water shows the main band at 410 nm (~max = 40000) and an inflection at 427 nm. The shape of the band is not symmetric and the lineshape is probably the envelope of two (or more) electronic components, whose absorption maxima are slightly separated. The corresponding lineshape of bilirubin is more regular and in this region the maximum, which is flatter than that of spin-labelled bilirubin, lies between 430 and 440 nm. It may be argued that the two electronic transitions associable to different conformations of bilirubin (see Ref. 26) have absorption maxima even closer than spin-labelled bilirubin. Binding to the protein was investigated through absorption spectra of spin-labelled bilirubin solutions conraining different amounts of human serum albumin. The visible absorption curves reported at different r values are shown in Fig. 4. The intensity of the inflection, present in the spectrum of the isolated spin-labelled bilirubin at ?~= 427 nm, increases starting from r = 5. At lower r values another absorption maximum, previously concealed under the most intense band, emerges at
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347
h = 450 nm. Moreover, the isosbestic point is clearly evidenced at h = 418 nm (c = 40800). This finding suggests that equilibrium between at least two forms of spin-labelled bilirubin may be established when human serum albumin is in excess. However, it is difficult to assess what really happens. The experimental curves do not often fit the isobestic point for r = 5, or in the borderline case in which human serum albumin is absent. This m a y be due to the degradation of spin-labelled bilirubin, more effective when human serum albumin is absent [27], or to the presence of a third species [26]. The electronic spectra of the bilirub i n / h u m a n serum albumin system show a corresponding variation in the lineshape (see Ref. 7), but in this case the isobestic point is absent.
Circular dichroism A solution of spin-labelled bilirubin in phosphate buffer does not exhibit a dichroic signal. The C D curve is observed in the region 360-600 nm when human serum albumin is added to the solution of spin-labelled bilirubin at p H 7.4 in phosphate buffer 0.1 M. At molar ratios r < 1 the shape of the curve is symmetric with a maximum at 425 nm, crossover point at 440 nm, and minim u m at 475 nm, as shown in Fig. 5 (Type II complex, according to K a m u s a k a et al. [28]). The C D sign is opposite with respect to the corresponding solution of b i l i r u b i n / h u m a n serum albumin system (Type I complex) [12,28]. It is inter-
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Fig. 6. Observed C D absorption spectra for the system spinlabelled bilirubin/bovine serum albumin at different spinlabelled bilirubin/bovine serum albumin values: 0.7; 1.0; 2.0; 5.0. Bovine serum albumin = 2.5.10 - 5 M; 0.1 M phosphate buffer at p H 7.4. Optical path length = 1 cm; gain = 5.10 -6.
esting to note that in the presence of bovine serum albumin the C D curve shows the same behaviour as observed for human serum albumin, with a maximum at 410 nm, crossover point at 431 nm, and a minimum at 468 nm. The spectra recorded at several r values are shown in Fig. 6. The values of Ac for the spin-labelled bilirubin/ h u m a n serum albumin system, referred to the total spin-labelled bilirubin concentration and related to the wavelengths at 425 and 475 nm, were measured at several r values and are shown in Fig. 7. The curves decrease regularly in the 0 < r < 2.0 = ~, = 425 nm
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Fig. 5. Observed C D absorption spectra for the system spinlabelled b i l i r u b i n / h u m a n serum albumin in 0.1 M phosphate buffer at p H 7.4; [human serum a l b u m i n ] = 2.5.10 -5 M; r = 0.1; 0.2; 0.3; 0.5; 1.0; optical path length = 1 cm; gain = 5- 10 -6.
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Fig. 8. Observed C D absorption for the system spin-labelled b i l i r u b i n / h u m a n serum albumin in 0.1 M phosphate buffer at different r values: 1.0; 2.0; 4.0 and 5.0. Spin-labelled bilirubin = 1.8.10 -5 M; optical path lenght = ! cm; gain = 2.10 -6.
range. For human serum albumin --* oo Ac goes to +31 ( ~ = 4 2 5 nm) and to - 3 0 ( ~ = 4 7 5 nm), respectively. The trend of Ac for bilirubin, obtained under the same experimental conditions, is qualitatively different, as the curves show a maxim u m at a ratio of r = 1.5, and then decrease regularly [29]. For larger r values the curves of the spin-labelled b i l i r u b i n / h u m a n serum albumin system are composite, as shown in Fig. 8. It is noteworthy that curve c in Fig. 9 (Xmax = 452 rim; ~o = 425 nm; X mi, = 400 nm), i.e., the difference between the two measured at r = 5 (curve a) and r = 1 (curve b), has the same sign as the corresponding one for the bilirubin/human serum albumin system measured under the same conditions of p H and buffer [12,28]. Some significant points are raised by the C D results. Firstly, the monotonic trend of the Acs measured for the spin-labelled bilirubin/human serum albumin system at low r values (below 1.5) suggests the existence of a CD active species whose sign is opposite to that of bilirubin; for larger r values a second active species appears, whose sign is similar to that of bilirubin. In bovine serum albumin the signs of the C D curves of complexes
Wavelength (nm)
Fig. 9. C D absorption for the system spin-labelled bilirubin/ h u m a n serum albumin (curve c) obtained by subtracting curve b (r = 1) from curve a (r = 5); [human serum albumin] = 2.5. 10 -~ M; 0.1 M phosphate buffer at pH 7.4; optical path length = l cm; gain = 5- 10 -6.
II and I (see Fig. 6) of spin-labelled bilirubin are similar to those measured for bilirubin at ratios of bilirubin/bovine serum albumin less than 2; it is noteworthy that for bilirubin at higher bilirubin/ bovine serum albumin ratios the curves change gradually, completely inverting the sign, with a crossover point at 437 nm [28]. The present state of knowledge of the signs of the dichroic curves for spin-labelled bilirubin and bilirubin is summarized in Table I. To determine whether the signs of the CD curves of the spin-labelled bilirubin/human serum albumin system could be related to protein conformational changes caused by the interaction, samples of human serum albumin partially saturated by spin-labelled bilirubin (r = 0.5 and 0.7) were measured after the addition of different quantities of bilirubin. When small quantities of bilirubin are added, the intensity of C D bands associated with spin-labelled bilirubin decreases, but when the concentration of bilirubin increases the sign of these CD bands change, as clearly evidenced in Fig. 10. To check the hypothesis that the CD
349
TABLE I STATE O F K N O W L E D G E O F T H E SIGNS O F C D C U R V E S F O R SPIN-LABELLED B I L I R U B I N ('SBR) A N D B I L I R U B I N (BR) IN H U M A N S E R U M A L B U M I N (HSA) A N D BOVINE S E R U M A L B U M I N (BSA) Data for spin-labelled bilirubin are from the present work; those for bilirubin are from Refs. 12 and 28, except for the second in bovine serum albumin, which is from Ref. 28 alone.
HSA
Site
SBR
1st
/% . "-ST-
BR •
Fx x/ •
2nd
1st
t'xk / •
/XX J
•
BSA 2nd
/'Xk . / .
•
" ~
a
From Ref. 28.
spectra of the spin-labelled bilirubin/human serum albumin/bilirubin ternary systems are the sum of two binary components, spin-labelled bilirubin/human serum albumin and bilirubin/human serum albumin, respectively, the ternary curve
was decomposed as shown in Fig. 11, curve c being obtained by subtracting curve b (spinlabelled bilirubin/human serum albumin) from a (spin-labelled bilirubin/human serum albumin/bilirubin). In the figure the CD curve for the bilirubin/human serum albumin binary system is shown for comparison. It should be noted that the difference curve has the same shape as that of the bilirubin/human serum albumin system, with a slight increase in intensity• Similar decompositions have been performed for other ternary curves and the addition rule was always checked. It is worthy of note that for spin-labelled bilirubin/human serum albumin = 0.8, i.e., when the high-affinity site is practically occupied, the ratio between the intensities at 457 nm of the 'difference curve' (bilirubin/human serum albumin, curve c in Fig. 11) and the corresponding curve measured for the binary system (curve d in Fig. 11) is practically constant and amounts to 1.6. This value corresponds to the intensity ratio between the CD curves ascribed by Beaven et al. [29] to the second and the first high-affinity sites of the bilirubin/human serum albumin system. This finding can be explained provided that we assume that in ternary systems either the rotatory power of bilirubin associated to the second high-affinity site is observed [28,29], or that spin-labelled biT
I
I
I C
AKo~ 60
b
20
-20
-6(] 360
400
440
480 520 Wavelength (nm)
Fig. 10. Observed C D absorption for the system spin-labelled b i l i r u b i n / h u m a n serum albumin ( 2 . 5 . 1 0 - S M ) / b i l i r u b i n in 0.1 M phosphate buffer at pH 7.4; spin-labelled bilirubin/hum a n serum albumin = 0.7; b i l i r u b i n / h u m a n serum albumin = 0.0; 0.05; 0.1; 0.3; 0.5; 0.7; 1.0. Optical path length = 1 cm; gain = 5.10 -6.
Wavelength (nm)
Fi 8. i l. C D absorption assigned for the system bflirubin/hum a n serum albumin (curve c ) o b t a i n e d by subtracting curve b (spin-labelled b i l l r u b i n / h u m a n serum a l b u m i n = 0 . 8 ) from curve a (spin-labelled b i l i r u b i n / h u m a n serum aibumin/bilirubin = 0 : 8 / I / 0 : 5 ) ; curve d represents the C D curve for the binary system b i l i r u b i n / h u m a n serum albumin = 0.5.
350 lirubin is in part displaced by bilirubin from the first high-affinity site and enters the second site. Thus the contribution of the Type I curve, ascribed to bilirubin only, according to the preceding interpretation, is increased, and that of the Type II curve due to spin-labelled bilirubin at the first site is reduced. Some comments on the nature of the binding of spin-labelled bilirubin to human serum albumin can be drawn. Firstly, the interaction occurs through the outer end of spin-labelled bilirubin not including the aliphatic chain, which probably remains outside the macromolecules, as evidenced by ESR spectra. This behaviour prevails in both human serum albumin and bovine serum albumin interactions. In other words, there are affinity sites in human serum albumin and bovine serum albumin inducing CD of the same sign, although one cannot assert that the sites are the same. Moreover, sites of lower affinity are present, and in this case CD with an opposite sign is detected, according to whether the binding occurs with human serum albumin or bovine serum albumin. Assuming that the CD sign may be associated with the way the micromolecule enters the macromolecule, the CD curves related to spin-labelled bilirubin/human serum albumin and spin-labelled bilirubin/bovine serum albumin, holding the same sign, suggest that spin-labelled bilirubin occupies the highest-affinity binding sites of human serum albumin and bovine serum albumin and enters in the same way. Conclusions
Spin-labelled bilirubin gives rise to three types of association with human serum albumin (or bovine serum albumin). Two correspond to binding constants of about 107 and 106 1. mol-1, respectively, and the third one corresponds to a binding of about 103 1. mol-1. The latter association, involving the spin-labelled arm, was evidenced by ESR spectroscopy. The two former associations come out clearly on titration of the albumin by fluorescence quenching and leave the main part of the spin-labelled aliphatic chain so free that its environment is the same as that of spin-labelled bilirubin dissolved in aqueous solution. Moreover, it appears from the difference CD
spectra that binding with the site of highest-affinity induces a double Cotton effect, positive at lower wavelengths and negative at higher wavelengths. In contrast, the site of the lowest affinity is associated with a nearly symmetrical Cotton effect with an opposite sign. The analogy between the two high-affinity associations of spin-labelled bilirubin and bilirubin with human serum albumin appears to be very pronounced. For example, the magnitudes of the binding constant measured by fluorescence quenching are very similar, and in both cases a CD bi-signed Cotton effect results. It is likely that the protein site involved in the association with spin-labelled bilirubin and bilirubin are roughly the same, minor conformational changes excepted. On the other hand this finding is understandable by considering the close structural analogies between bilirubin and spin-labelled bilirubin, in which the bilirubin skeleton is left untouched, and a steric change of the side chain at the C-18 position occurs. This slight conformational difference of the chain at the distal position of the tetrapyrrolic moiety can lead to inversion of the Cotton effect (related to the high-affinity site) observed in the interaction with human serum albumin (but not with bovine serum albumin) by going from bilirubin to spin-labelled bilirubin. This is probably due to a reciprocal fitting of the conformations of the ligand and the protein which, in the thermodynamically favoured location, involves two conformations of opposite chirality for bilirubin and spin-labelled bilirubin. Conformational changes of the protein reflecting on the induced chirality of the ligand are known in the literature [30], and in particular for the bilirubin/ human serum albumin system, the bi-signed Cotton effect proved to be inverted by going from neutral to acid pH [12,28]. If, as it seems, spinlabelled bilirubin enters the sites of the albumin like the molecule of the natural pigment, the following conclusion may be drawn from the measurements reported on the spin-labelled bilirubin/ human serum albumin and bilirubin/human serum albumin complexes. In the interaction between bilirubin and human serum albumin the side chain C-18 is not directly involved in the binding, and this finding suggests that bilirubin enters the affinity sites of human serum albumin through deep insertion of only one of the two
351
extreme tings (or one pyrromethenone moiety), leaving the ring (or half molecule) free to originate a molecular conformation compatible both with its actual steric hindrance (different in bilirubin and spin-labelled bilirubin) and with the conformation preferred by the site, which is different for human serum albumin and bovine serum albumin and depends on pH. This is consistent with the changes of the Cotton effect sign, and could occur with the conformation of a tetrapyrrolic molecule either of the ridge-tile type (as in solid bilirubin or CHL13 solution [31,17]) or the helix type [32]. References 1 Brown, J.R. (1977) in Albumin Structure, Function and Uses (Rosender, V.M., Oratz, M., and Rothschild, M.A., eds.), pp. 27-52, Pergamon Press, New York 2 McMenamy, R.H. (1977) in Albumin Structure, Function and Uses (Rosender, V.M., Oratz, M. and Rothschild, M.A., eds.), pp. 143-158, Pergamon Press, New York 3 Arias, I.M. and Jansen, P. (1975) in Jaundice (Goresky, C.A. and Fischer, M.M., eds.), pp. 175-188, Plenum Press, New York 4 Schmid, R. (1975) Bilirubin: in Jaundice (Goresky, C.A. and Fischer, M.M., eds.), pp. 43-55, Plenum Press, New York 5 Scheidt, P.C., Mellitz, E.D., Hardy, J.B., Drage, J.S. and Boggs, T.R. (1977) J. Pediatr. 91,292-297 6 McDonagh, A.F. (1979) in The Porphyrins (Dolphin, D., eds.), pp. 294-491, Academic Press, New York 7 Brodersen, R. (1979) CRC Crit. Rev. Chem. Lab. Sci. 11, 305-399 8 Hsia, J.C., Kwan, N.N., Er, S.S., Wood, D.J. and Chance, G.W. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1542-1545 9 Wood, D.J. and Hsia, J.C. (1977) Biochem. Biophys. Res. Commun. 76, 863-868 10 Manitto, P. and Monti, D. (1973) Experientia 29, 137-138 11 Onishi, S., Kawade, N., Iton, S., Isobe, K. and Sugiyama, S. (1980) Biochem. J. 190, 527-532 12 Blauer, G., Harmatz, D. and Snir, J. (1972) Biochim. Biophys. Acta 278, 68-88
13 Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K.M. (1969) p. 490, Data for Biochemical Research, Clarendon Press, Oxford 14 Hampton, A., Slotin, L.A. and Chawla, R.R., (1976) J. Med. Chem. 19, 1279 15 McConnel, H.M., Deal, W. and Ogata, R.T. (1969) Biochemistry 8, 2580-2585 16 Levine, R.L. (1977) Clin. Chem. 23, 2292-2301 17 Manitto, P. and Monti, D. (1976) J.C.S. Chem. Commun. 122-123 18 Mugnoli, A., Manitto, P. and Monti, D. (1978) Nature 273, 568-569 19 Berde, C.B., Mudson, B.S., Simoni, R.D. and Skar, L.A. (1979) J. Biol. Chem. 254, 391-400 20 Klotz, I.M. and Hunston, D.L. (1971) Biochemistry 10, 3065-3069 21 Barzaghi, M.B., Gamba, A., Morosi, G. and Simonetta, M. (1978) J. Phys. Chem. 82, 2105-2114 22 Nordio, P.L. (1975) in Spin-Labeling, Theory and Applications (Berliner, L., ed.), pp. 5-52, Academic Press, New York 23 Freed, J.H. (1976) in Spin-Labeling, Theory and Applications (Berliner, L., ed.), pp. 53-132, Academic Press, New York 24 Reddoch, A.M. and Konishi, S. (1979) J. Chem. Phys. 70, 2121-2130 25 Chen, R.F. (1973) in Fluorescence techniques in cell biology (Thaen, A.A. and Serntz, M., eds.), pp. 273-282, SpringerVerlag, Berhn 26 Holzwarth, A.R., Langer, E., Lehner, H. and Scbaffner, K. (1980) Photochem. Photobiol. 32, 17-26 27 Witt, T.K. (1968) Bile Pigments, p. 270, Academic Press, New York 28 Kamisaka, K., Listowsky, I., Betheil, J.J. and Arias, I.M. (1974) Biochim. Biophys. Acta 365, 169-180 29 Beaven, G.H., D'Albis, A. and Gratze, W.B. (1973) Eur. J. Biochem. 33, 500-510 30 Branca, M. and Pispisa, B. (1977) J. Chem. Soc. Faraday I, 73, 213-229 31 Bonnet, R., Davis, J.E. and Hursthouse, M.B. (1976) Nature (London) 262, 326-328 32 Blauer, G. and Wagni~re, G. (1975) J. Am. Chem. Soc. 97, 1949-1954
341
BBA 31444
THE BINDING OF BILIRUBIN TO ALBUMIN
A STUDY USING SPIN-LABELLED BILIRUBIN M A R I O B K A N C A a A L D O G A M B A a.,, PAOLO M A N I T T O b, D I E G O M O N T I c and G I O V A N N A S P E R A N Z A b
a Istituto di Chimica Fisica, Universit,~ di Sassari, V. Vienna 2, 07100 Sassari, b lstituto di Chimica Organica, Universiti~ di Milano, and ¢ Centro per 1o Studio delle Sostanze Organiche Naturali del CNR, Milano (Italy) (Received September 22nd, 1982)
Key words: Spin label," Bilirubin; Protein binding," Human serum albumin; Cotton effect
Binding between human serum albumin and a spin-labelled derivative of bilirubin was investigated by circular dichroism, fluorescence quenching, electron spin resonance and visible spectroscopy. The orders of magnitude of the binding constants obtained by flurorescence quenching and electron spin resonance spectroscopies were 10 7 and 10 a l - m o l - 1, respectively. These data suggest that most spin-labelled bilirubin interacts with human serum albumin at the side not holding the spin-labelled side-arm. CD measurements showed the presence of at least two sites, associated with opposite Cotton effects. It is worthy of note that the Cotton sign of the first site is inverted with respect to the corresponding one of bilirubin. CD measurements on mixed systems (spin-labelled bilirubin/human serum albumin/bilirubin) were also performed. The decomposition of the ternary curves shows that the rotatory power of bilirubin bound to human serum albumin is higher in the ternary system than in the binary (bilirubin/human serum albumin). The corresponding CD measurements for the binding between spin-labelled bilirubin and bovine serum albumin are also reported and discussed.
Introduction
Interactions of bilirubin, the end product of heme catabolism with serum albumin, the most abundant protein in mammalian plasma, have been extensively studied for both theoretical interest and clinical importance [1-5]. Our interest in bile pigment metabolism led us to study the interaction between bilirubin and proteins using suitably synthesized spin-labelled bilirubins. To our knowledge no investigation of such a kind has been reported in the literature, although a great deal of work of different origin has been carried out by means of physical, spec* To whom correspondence should be addressed. 0167-4838/83/0000-0000/$03.00 © 1983 Elsevier Biomedical Press
troscopic and chemical methods [6,7]. The only spin-labelled molecule reported to be bound to human serum albumin at the highest affinity site for bilirubin is 1-N-(2,2,6,6-tetramethyl-l-oxyl-4piperidinyl)-5-N-(1-aspartate)-2,4-dinitrobenzene [8], i.e., a completely different ligand from bilirubin. Like bilirubin the compound appears to be a suitable clinical tool for determining the concentration of vacant bilirubin binding sites of albumin in serum [9]. We present here the first synthesis of a spinlabelled bilirubin (6) and the specific binding of it to human serum albumin. The structure of spinlabelled bilirubin was planned so that one of the two outer pyrromethenone moieties would have a spin label, thus allowing the other one (and most
342 of the tetrapyrrolic skeleton) to interact with the protein, as is the case with bilirubin binding. Materials and Methods
Bilirubin. Bilirubin (Merck) was purified as previously described [6], and checked for purity by TLC [10] and HPLC [11], giving c = 60700 at 454 nm [6] in CHC13. 4-Amino-2,2,6,6-tetramethylpiperidin- 1-yloxy (3) was obtained from Aldrich. Human serum albumin. Essentially fatty acidfree human serum albumin (Sigma No. A-1887) was used in all experiments. Molar protein concentrations were based on weighing and on a molecular weight of 69000 [1] ( c = 3 6 5 0 0 at 278-279 nm, 25°C, pH 7.4) [12]. Deionized water was redistilled. A 0.1 M phosphate buffer, pH 7.4, was prepared according to Clark and Lubs [13] and used to obtain stock albumin solutions (10 mg/ml, approx. 1.5 • 10 -4 M). These were kept at 0-5°C for a period of time not exceeding 1 week (no significant change of c was observed). Bovine serum albumin. Bovine serum albumin from Merck (Art. 12018) was used, without further purification, as described above for human serum albumin (bovine serum albumin, ~ = 47 000 at 278-279 nm, 25°C, pH 7.4) [12]. Preparation and measurements of solutions. Stock solutions of bilirubin and spin-labelled bilirubin was prepared and used immediately. About 2.5 mg of the pigment were dissolved in 0.25 ml of 0.02 M aqueous NaOH, diluted to 25 ml with distilled water (1.5 • 10 - 4 M ) . The solutions were protected form light during preparation. Exact solute concentrations were determined spectrophotometrically in the presence of an almost equimolecular quantity of human serum albumin at pH 7.4 in 0.1 M phosphate buffer (bilirubin: c = 4 6 5 0 0 at 458 nm; spin-labelled bilirubin: c = 40 800 at 418 nm). Unless stated otherwise, a measured amount of the bilirubin stock solutions was added to a protein solution with stirring, followed by dilution with phosphate buffer (0.1 M) or distilled water, in order to obtain the concentrations and molar ratios between the ligand and the protein desired. Usually the pH of the final solutions was within the range of 7-8. It was adjusted to the value of 7.4 by addition of a few drops of 1 M NaOH or KH2PO 4.
Solutions were protected from light until measurement. Synthesis of spin-labelled bilirubin (_6). 1,3-Propandithiol (0.25 ml) was added to a solution of bilirubin (100 mg) in CHCI 3 (100 ml). After the addition of a few crystals of p-toluensulphonic acid the reaction mixture was stored overnight at room temperature in the dark. Evaporation of the solvent under reduced pressure to 10 ml, followed by addition of MeOH, led to a precipitate which was filtered, washed twice with MeOH and dried. The solid (77 mg) was shown to be pure (2) by TLC (moving more slowly than bilirubin on silica gel plates, benzene/CHC13/MeOH(53 : 45 : 2)) and elemental analysis (calc. for C 3 6 H 4 4 N 4 0 6 S 2 : C, 62.42; H, 6.35; N, 8.00; found: C, 62.34; H, 6.32; N, 7.89). nH-NMR (C2HC13; TMS, 8: 1; 53 d (3H, J = 8 Hz, Me-CH(S-)-C(18)); 1.97s (3H, Me-C(2)); 2, 15s (9H, 3Me); 2.5-3.1m (propionic acid and C(18') side chains); 3.96q (1H, J = 8Hz; Me-CH-(S-)-C(18)); 4.06s (2H, central CH2-); 5.4-5.7rn (CH 2 = CH-); 6.11s and 6.19s (methine bridges); 6.4-6.8m (CH 2 = CH-); 9.26s (2H, pyrole NHs); 10.69s and 10.77s (2H, amide NHs). N-Iodoacetoxylsuccinimide (_4) was prepared according to the procedure of Hampton et al. [14] in 60% yield, m.p. 148°C (literature [14], 148-150°C; calc. for CrHrlN404: C, 25.44; H, 2.14; N, 4.95; found: C, 26.55; H, 2.36; N, 5.20). nH-NMR (DMSO-d 6, TMS, 8): 2.81s (4H); 4.1s (2H). N-(2,2,6,6-Tetramethyl-4-piperidin- 1yloxy)iodoacetamide (_5) was obtained by adding compound (4) (1 g dissolved in 20 ml H4furan and 3 ml phosphate buffer [13], pH 7.5) to a solution of 4-amino-2,2,6,6-tetramethylpiperidin- 1-yloxy (3) (500 mg) in 10 ml H j u r a n . After 30 min stirring at room temperature in N 2 atmosphere, the reaction mixture was extracted with CHCI 3 and the organic phase washed with dilute HCI, and dried on anhydrous sodium sulphate. Evaporation of the solvent under reduced pressure gave a red oil, which was chromatographed on silica gel column eluting with 1% MeOH in CHCI 3. Fractions containing pure (5), as shown by TLC (silica gel plates, C H C l J E t O H 5 : 1), were evaporated and the residue crystallized from benzene, affording dark orange needles (m.p. 113-116°C, lit. 114-117°C [15]); (calc. for C 1nH20IN202: C, 38.95; H, 5.94; N, 8.26; found: C, 39.0; H, 5.8; N, 7.9).
343 To obtain the spin-labelled bilirubin (6) the SHbearing bilirubin (_2) (30 mg in a mixture of 10 ml N, N'-dimethylformamide and 1.5 ml 0.1 M phosphate buffer, pH 7.8) was treated with compound (5) (20 mg in 0.5 ml dimethylformamide). After 1 h at room temperature under a slow stream of argon the reaction mixture was poured into water (20 ml) and extracted with CHC13 (25 ml) after the addition of an excess of solid carbon dioxide. The organic phase was then dried on anhydrous sodium sulphate and evaporated to a small volume (approx. 0.5 ml) in a vacuum. The precipitate resulting from the addition of methanol was collected (25 mg) and shown to be spin-labelled bilirubin (6) by 1H-NMR (C2HC13, TMS, 8): 1.53 d (3H, J = 8 Hz, Me-CH(S-)-C(18)); 1.99s (3H,MeC(2); 2.16s (3Me); 4.05s (central bridge largely hiding the quartet due to Me-CH-(S-)-C(18)); 5.4-5.7m (CHE=CH-); 6.14s and 6.21s (two singlets due to the methine bridges); 6.4-6.8m (CH 2 = CH-); 9.285s, (pyrrole NHs); 10s and 10.80s (amide NHs); all the protons C-H present in the C(18') side-chain gave rise to very broad peaks buried under the sharp signals of the biladiene substituents in the range of 1.3-3.3. The purity of (6) used in spectroscopic measurements was checked by TLC (benzene/CHC13/MeOH (45 : 45 : 10)) and elemental analysis. Its ESR spectrum in phosphate buffer, pH 7.4, is displayed in Fig. 2(a). Elemental analysis calc. for C 4 7 H 6 3 N 6 0 8 S 2 : C , 62.43; H, 7.02; N, 9.29; found: C, 62.11; H, 6.88; N, 9.15. ESR. The experimental samples (70 #l) were prepared by combining stock solutions of 5. 10 -4 M human serum albumin (0.1 M phosphate buffer, pH 7.1), and 1 • l 0 - 2 M spin-labelled bilirubin (0.5 M NaOH) with a micropipette (50 and 10 #l). The final pH values of the solutions were 7.4-7.5; after mixing the samples were transferred by capillarity into a disposable pipette (Coming) which was used for ESR measurements. The concentration of the spin-labelled bilirubin, the spin-labelled arm of which is free in solution, was proportional to the height of the high-field peak of the sharp triplet. Since the total amount of spin-labelled bilirubin was known, the amount of spin-labelled bilirubin bound to serum albumin could be determined. Absorption spectroscopy. The experimental sam-
pies held about 1 • 10-5 M concentration of spinlabelled bilirubin, or bilirubin and variable concentrations of human serum albumin. The spectra were recorded in the range 350-550 nm with a Varian 634 spectrophotometer. Fluorescence quenching. The measurements were performed with an Aminco-Bowman spectrofluorimeter. The work-exciting wavelength was 294 nm and that of emission 340 nm. Three cuvettes were used in the experiment. One, containing phosphate buffer (0.1 M, pH 7.4), was used as zero percent reference; the second one, containing 2.5 • 10 -6 M human serum albumin was used as 100% fluorescence, and the third one holding exactly 2 ml human serum albumin was that used for the titrating solution. A solution of spin-labelled bilirubin (or bilirubin), at a concentration 20-times greater than that of the human serum albumin was added to the third cuvette. Then the fluorescence of the three solutions was measured and the procedure was repeated at least twenty times after adding 10 #1 of the spin-labelled bilirubin (or bilirubin) solution. All the measurements were corrected for the inner filter effect, and for dilution. Binding equilibria were analyzed by the method of Levine [16]. Circular dichroism. The experimental samples held 2.5.10-SM concentrations of serum albumin and variable concentrations of spin-labelled bilirubin and bilirubin. The spectra were recorded in the 350-700 nm range with a Dicrograph Mark III Jobin-Yvon. Cells of 1 or 0.1 cm path-length were employed. Results and Discussion'
Spin-labelled bilirubin (6) was obtained in satisfactory yields according to the convergent synthesis shown in Scheme I. The functionalization of the spin-labelled bilirubin side-chain at the 18 position was achieved taking advantage of the known acid-catalyzed regiospecific addition of nucleophils to an exo-vinyl group [10]. The structure of spin-labelled bilirubin was affirmed by its NMR spectrum, which also indicated strong conformational analogies between spin-labelled bilirubin and bilirubin in solution. Thus, the spinbearing side-arm in spin labelled bilirubin appears to have only slight effect on the conformational
344
HOOC COOH
2.8
I
I
I
I
1/~
v
H
H
H
H
(1)
2.4
O (3)
2.0 1.6
HS-ICH2lfSH
1 ~
NH-CO-CH2-1
S-(CH213"-SH
(~NI%0
~,~~
(2)
(5) I
I
I
I
4
8
12
16
20
10-6/cf
~I
HOOC COOH " O ~
18
S-(CH2)3"S-CH2-CO-I~,H
(6) SCHEME
Fig. 1. Klotz's double reciprocal binding plot for titration of human serum albumin (2.10 .6 tool-1 -I) with spin-labelled bilirubin(e) or bilimbin(~.) by fluorescencequenching. ? is the number of moles of spin-labelled bilirubin or bihrubin bound/mole human serum albumin. Cr is the number of moles of spin-labelledbilirubin or bilirubin which are free in solution.
O I
stabilization of the a , c - b i l a d i e n e skeleton, which c a n a s s u m e the ridge-tile form typical of bilirubin-like c o m p o u n d s [17,18].
K 2 = 1.3- 106 1. m o l - ] , n 2 = 1.62 for spin-labelled bilirubin, a n d K 2 = 6.7- 106 1 • mol - I , n 2 = 1.19 for bilirubin, were o b t a i n e d p e r f o r m i n g a titration at a h u m a n serum a l b u m i n c o n c e n t r a t i o n higher t h a n ( 1 - 1 . 5 ) . 10 -5 M. U n d e r these c o n d i t i o n s es-
Fluorescence quenching The technique employed, which allows det e r m i n a t i o n of both b i n d i n g affinity a n d b i n d i n g capacity, is described in the Materials a n d Methods section. Even though the b i n d i n g constants o b t a i n e d from fluorescence q u e n c h i n g for the complexes of b i l i r u b i n with h u m a n serum a l b u m i n are easily available in the literature [19], it has been explicitly reported that the reproducibility of the m e a s u r e m e n t s was poor [16]. So, within the scope of a significant c o m p a r i s o n between the b i n d i n g of spin-labelled b i l i r u b i n a n d b i l i r u b i n u n d e r c o m p a r a b l e conditions, the fluorescence q u e n c h i n g for the b i l i r u b i n / h u m a n serum alb u m i n system was re-measured. The reciprocal b i n d i n g plots of Fig. 1 show the existence of two sites at least. According to Klotz a n d H u n s t e n [20], the following parameters were o b t a i n e d : K I = 2 . 8 7 . 1 0 7 1 • mo1-1, n 1 = 1, for spin-labelled bilirubin; K l = 1.26.107 1 • m o l - l, nl = 0.94, for bilirubin. The parameters relating to the second site,
J
(P
J
S
(q
f
i (~
Fig. 2. ESR spectra for (a) spin-labelled bilirubin in 0.1 M phosphate buffer at pH 7.4; (b) human serum albumin added to solution (a) at r = 0.4; (c) solution (a) frozen at - 18°C; (d) frozen solution (-18°C) of spin-labelled bilirubin in 0.1 M phosphate buffer at pH 7.4; r = 2.85; r = spin-labelled bilirubin/human serum albumin.
345
sentially no unbound bilirubin is detectable until the first site is saturated [16]. Within the limits of the type of interaction detectable by the present technique, it appears that the bindings of spinlabelled bilirubin and bilirubin to human serum albumin are comparable. However, on the basis of the data obtained from the fluorescence measurements reported in Fig. 1, we cannot assert that spin-labelled bilirubin and bilirubin occupy the same site of high affinity of human serum albumin, a n d / o r in the same manner. By considering that the interaction is very similar one cannot exclude the possibility either that the two sites for bilirubin and spin-labelled bilirubin are very close (i.e., at a comparable distance from tryptophan), or that the same site is differently engaged. Electron spin resonance
A representative spectrum of a solution of a spin-labelled bilirubin in phosphate buffer at pH 7.4 is shown in Fig. 2(a). The hyperfine pattern is typical of a nitroxide spin label, and a moderate anisotropy of the hyperfine components is evident in the spectrum. Simulation through a least-square
1/'1) 16
x
0
'
Jo
'
,~
'
lo-3/cf
&
Fig. 3. ESR measurements. Klotz's double reciprocal binding plot of spin-labelled bilirubin to h u m a n serum albumin. Different symbols refer to different sets of measurements; ~ is the n u m b e r of moles of spin-labelled bilirubin b o u n d / m o l e h u m a n serum albumin. Cf is the number of moles of spin-labelled bilirubin which are free in solution.
fitting [21] gives the following spectral parameters: the nitrogen hyperfine splitting a N(N°)= 17.18 + 0.04 G; the linewidth W = 1.67 + 0.08 G, and a value of 0.13 G for B(N), a linewidth parameter [22] describing the mobility of the spin label. Simulation of the spectrum of a sample of the same free spin label gives the following spectral parameters: a N(NO) = 17.90 + 0.02 G, W = 1.42 + 0.05 G, B(N) = 0.045 + 0.027 G. It is interesting to observe that the mobility in the same solvent is reduced by about three times when the label is bound to bilirubin. The ESR spectrum measured in the same solution in the presence of human serum albumin (r = spin labelled bilirubin/human serum albumin = 0.4) is shown in Fig. 2(b). The spectrum is qualitatively different, since two hyperfine splitting patterns, one typical of a bound fraction and the other associated with a more mobile species, can be observed. The magnitude of the splitting of the nitrogen belonging to the bound fraction is 2T~z = 69 G. A rought measurement of correlation time "r, according to a technique proposed by Freed [23], gives "r --- 2 . 1 0 -8 s. It appears that the spin-labelled chain is not completely immobilized. The hyperfine splitting constant of the nitrogen of the weakly bound fraction is identical (a N(N°) = 17,18 G) to the corresponding one of spin-labelled bilirubin in phosphate buffer solution in the absence of human serum albumin. The coincidence is significant and proves that the spin label either belongs to a fraction not implied in the binding with human serum albumin, or is in the oxydipyrromethene moiety of spin-labelled bilirubin interacting with human serum albumin faced to the water phase. As a matter of fact it is well known that the magnitude of the hyperfine splitting constant of the nitrogen of the nitroxide is strongly dependent on the dielectric constant and on other physical properties of the environment (see for example Ref. 24). The ESR spectrum of spin-labelled bilirubin in the glass phase recorded at - 1 8 ° C in phosphate buffer is shown in Fig. 2(c). The spectrum measured under the same conditions after the addition of human serum albumin to the solution (r = 2.85) is reported in Fig. 2(d). The upper spectrum (c) in Fig. 2 is broadened by spin-spin interactions, which depend on spin concentration, probably due to the
346
formation in solution of micelles of spin-labelled bilirubin [16]. The addition of human serum albumin probably leads to the breaking of its colloidal structure, so that spin-spin interactions responsible for the broadening are greatly reduced. The amount of spin label, free plus weakly bound to the macromolecule, was obtained through the calibration of the high-field peak of the sharp triplet [9]. The amount of strongly bound component was obtained by difference. According to the technique proposed by Wood and Hsia [9], the double reciprocal binding plot described by Klotz and Hunston [20] was built up using a series of ESR spectra measured at different values of r. The experimental points and the plot are shown in Fig. 3. The derived binding constant, which involves only the spin-label moiety, is 6.6-10 3 l" tool -1, the number of sites being n = 1. It is noteworthy that the presence of bilirubin (bilirubin/spin-labelled bilirubin/human serum albumin = 1 : 1 : 1) does not modify the magnitude of the binding constant of spin-labelled bilirubin to human serum albumin. This binding is lower by about four orders of magnitude that that derived from fluorescence quenching (see also Ref. 25). To correlate the results obtained by fluorescence and ESR measurements it has to be considered that the interaction is governed by an equilibrium process (Kn,or = 2.87" 1 0 7 1 • mol -I, K E S R ---- 6.6 • 1 0 3 1 • m o l - l) and that significantly different amounts of spin-labelled bilirubin and human serum albumin (fluor: 10 -6 M; ESR: 10 -4 M) were necessary to obtain the best answers by the two spectroscopic techniques. The observed difference of the K equilibrium constants has to be ascribed to the different quality of bound component detectable by the two spectroscopic techniques. ESR detects only the part of spin-labelled bilirubin whose spin-labelled arm is blocked, but cannot distinguish between spin-labelled bilirubin interacting with human serum albumin through the unsubstituted side and the free spin-labelled bilirubin. Fluorescence detects the total bound component. For example, at concentrations of human serum albumin = spinlabelled bilirubin = 1 0 - 6 mol- 1 - 1, it emerges from Knuor that 80% of spin-labelled bilirubin is bound, whereas from K E S R only 0.5% of spin-labelled bilirubin appears bound. In view of this it is easy
to deduce that most of the spin-labelled bilirubin interacts with human serum albumin through the side without the spin-labelled chain.
Visible absorption The electronic spectrum of spin-labelled bilirubin in water shows the main band at 410 nm (~max = 40000) and an inflection at 427 nm. The shape of the band is not symmetric and the lineshape is probably the envelope of two (or more) electronic components, whose absorption maxima are slightly separated. The corresponding lineshape of bilirubin is more regular and in this region the maximum, which is flatter than that of spin-labelled bilirubin, lies between 430 and 440 nm. It may be argued that the two electronic transitions associable to different conformations of bilirubin (see Ref. 26) have absorption maxima even closer than spin-labelled bilirubin. Binding to the protein was investigated through absorption spectra of spin-labelled bilirubin solutions conraining different amounts of human serum albumin. The visible absorption curves reported at different r values are shown in Fig. 4. The intensity of the inflection, present in the spectrum of the isolated spin-labelled bilirubin at ?~= 427 nm, increases starting from r = 5. At lower r values another absorption maximum, previously concealed under the most intense band, emerges at
i
i
i
5 6.10-4
4
3
2 5
0
350
400
450
500
550
Wavelength(nm) Fi 8. 4. A b s o r p t i o n s p e c t r a o f s p i n - l a b e l l e d b i l i r u b i n at d i f f e r e n t ratios of spin-labelled bilirubin/human serum albumin (r); s p i n - l a b e l l e d b i l i r u b i n = 1 . 1 8 . 1 0 - 5 m o l . l - I in 0.1 M p h o s p h a t e b u f f e r a t p H 7.4. (1) r = ~ ; (2) r = 3.19; (3) r = 1.29; (4) r = 0.64; (5) r = 0.27.
347
h = 450 nm. Moreover, the isosbestic point is clearly evidenced at h = 418 nm (c = 40800). This finding suggests that equilibrium between at least two forms of spin-labelled bilirubin may be established when human serum albumin is in excess. However, it is difficult to assess what really happens. The experimental curves do not often fit the isobestic point for r = 5, or in the borderline case in which human serum albumin is absent. This m a y be due to the degradation of spin-labelled bilirubin, more effective when human serum albumin is absent [27], or to the presence of a third species [26]. The electronic spectra of the bilirub i n / h u m a n serum albumin system show a corresponding variation in the lineshape (see Ref. 7), but in this case the isobestic point is absent.
Circular dichroism A solution of spin-labelled bilirubin in phosphate buffer does not exhibit a dichroic signal. The C D curve is observed in the region 360-600 nm when human serum albumin is added to the solution of spin-labelled bilirubin at p H 7.4 in phosphate buffer 0.1 M. At molar ratios r < 1 the shape of the curve is symmetric with a maximum at 425 nm, crossover point at 440 nm, and minim u m at 475 nm, as shown in Fig. 5 (Type II complex, according to K a m u s a k a et al. [28]). The C D sign is opposite with respect to the corresponding solution of b i l i r u b i n / h u m a n serum albumin system (Type I complex) [12,28]. It is inter-
,, "~l~
'
~o
~o
'
,Io
'
'
~o
5~0
Fig. 6. Observed C D absorption spectra for the system spinlabelled bilirubin/bovine serum albumin at different spinlabelled bilirubin/bovine serum albumin values: 0.7; 1.0; 2.0; 5.0. Bovine serum albumin = 2.5.10 - 5 M; 0.1 M phosphate buffer at p H 7.4. Optical path length = 1 cm; gain = 5.10 -6.
esting to note that in the presence of bovine serum albumin the C D curve shows the same behaviour as observed for human serum albumin, with a maximum at 410 nm, crossover point at 431 nm, and a minimum at 468 nm. The spectra recorded at several r values are shown in Fig. 6. The values of Ac for the spin-labelled bilirubin/ h u m a n serum albumin system, referred to the total spin-labelled bilirubin concentration and related to the wavelengths at 425 and 475 nm, were measured at several r values and are shown in Fig. 7. The curves decrease regularly in the 0 < r < 2.0 = ~, = 425 nm
30
~,Kob !
"
Wavelength (nm)
, 6O
'
20
I0
2(
o i
t
i
i
,
Q9
!~3
1!7
-10
-20
~,=47§nm -60
~o
,~o
,~o
~o
~o
Wavelength (nm)
Fig. 5. Observed C D absorption spectra for the system spinlabelled b i l i r u b i n / h u m a n serum albumin in 0.1 M phosphate buffer at p H 7.4; [human serum a l b u m i n ] = 2.5.10 -5 M; r = 0.1; 0.2; 0.3; 0.5; 1.0; optical path length = 1 cm; gain = 5- 10 -6.
dl
d5
21
23 [SBR/HSA]
Fig. 7. Dependence of A( values ( l . m o l . c m - n ) at 425 n m (upper curve) and 475 n m (lower curve) at p H 7.4 in 0.1 M phosphate buffer, vs. the molar ratio spin-labelled bilirub i n / h u m a n serum albumin. A( values are referred to the total spin-labelled bilirubin concentration.
348 10
i
f
i
i
a%,
a%b
i
i
100
6O 1.0 b
2O
-20
-6 -60 a
-10
,oo
,,Jo
1.0 I
48O
52O
-1~
Wavelength (nm)
Fig. 8. Observed C D absorption for the system spin-labelled b i l i r u b i n / h u m a n serum albumin in 0.1 M phosphate buffer at different r values: 1.0; 2.0; 4.0 and 5.0. Spin-labelled bilirubin = 1.8.10 -5 M; optical path lenght = ! cm; gain = 2.10 -6.
range. For human serum albumin --* oo Ac goes to +31 ( ~ = 4 2 5 nm) and to - 3 0 ( ~ = 4 7 5 nm), respectively. The trend of Ac for bilirubin, obtained under the same experimental conditions, is qualitatively different, as the curves show a maxim u m at a ratio of r = 1.5, and then decrease regularly [29]. For larger r values the curves of the spin-labelled b i l i r u b i n / h u m a n serum albumin system are composite, as shown in Fig. 8. It is noteworthy that curve c in Fig. 9 (Xmax = 452 rim; ~o = 425 nm; X mi, = 400 nm), i.e., the difference between the two measured at r = 5 (curve a) and r = 1 (curve b), has the same sign as the corresponding one for the bilirubin/human serum albumin system measured under the same conditions of p H and buffer [12,28]. Some significant points are raised by the C D results. Firstly, the monotonic trend of the Acs measured for the spin-labelled bilirubin/human serum albumin system at low r values (below 1.5) suggests the existence of a CD active species whose sign is opposite to that of bilirubin; for larger r values a second active species appears, whose sign is similar to that of bilirubin. In bovine serum albumin the signs of the C D curves of complexes
Wavelength (nm)
Fig. 9. C D absorption for the system spin-labelled bilirubin/ h u m a n serum albumin (curve c) obtained by subtracting curve b (r = 1) from curve a (r = 5); [human serum albumin] = 2.5. 10 -~ M; 0.1 M phosphate buffer at pH 7.4; optical path length = l cm; gain = 5- 10 -6.
II and I (see Fig. 6) of spin-labelled bilirubin are similar to those measured for bilirubin at ratios of bilirubin/bovine serum albumin less than 2; it is noteworthy that for bilirubin at higher bilirubin/ bovine serum albumin ratios the curves change gradually, completely inverting the sign, with a crossover point at 437 nm [28]. The present state of knowledge of the signs of the dichroic curves for spin-labelled bilirubin and bilirubin is summarized in Table I. To determine whether the signs of the CD curves of the spin-labelled bilirubin/human serum albumin system could be related to protein conformational changes caused by the interaction, samples of human serum albumin partially saturated by spin-labelled bilirubin (r = 0.5 and 0.7) were measured after the addition of different quantities of bilirubin. When small quantities of bilirubin are added, the intensity of C D bands associated with spin-labelled bilirubin decreases, but when the concentration of bilirubin increases the sign of these CD bands change, as clearly evidenced in Fig. 10. To check the hypothesis that the CD
349
TABLE I STATE O F K N O W L E D G E O F T H E SIGNS O F C D C U R V E S F O R SPIN-LABELLED B I L I R U B I N ('SBR) A N D B I L I R U B I N (BR) IN H U M A N S E R U M A L B U M I N (HSA) A N D BOVINE S E R U M A L B U M I N (BSA) Data for spin-labelled bilirubin are from the present work; those for bilirubin are from Refs. 12 and 28, except for the second in bovine serum albumin, which is from Ref. 28 alone.
HSA
Site
SBR
1st
/% . "-ST-
BR •
Fx x/ •
2nd
1st
t'xk / •
/XX J
•
BSA 2nd
/'Xk . / .
•
" ~
a
From Ref. 28.
spectra of the spin-labelled bilirubin/human serum albumin/bilirubin ternary systems are the sum of two binary components, spin-labelled bilirubin/human serum albumin and bilirubin/human serum albumin, respectively, the ternary curve
was decomposed as shown in Fig. 11, curve c being obtained by subtracting curve b (spinlabelled bilirubin/human serum albumin) from a (spin-labelled bilirubin/human serum albumin/bilirubin). In the figure the CD curve for the bilirubin/human serum albumin binary system is shown for comparison. It should be noted that the difference curve has the same shape as that of the bilirubin/human serum albumin system, with a slight increase in intensity• Similar decompositions have been performed for other ternary curves and the addition rule was always checked. It is worthy of note that for spin-labelled bilirubin/human serum albumin = 0.8, i.e., when the high-affinity site is practically occupied, the ratio between the intensities at 457 nm of the 'difference curve' (bilirubin/human serum albumin, curve c in Fig. 11) and the corresponding curve measured for the binary system (curve d in Fig. 11) is practically constant and amounts to 1.6. This value corresponds to the intensity ratio between the CD curves ascribed by Beaven et al. [29] to the second and the first high-affinity sites of the bilirubin/human serum albumin system. This finding can be explained provided that we assume that in ternary systems either the rotatory power of bilirubin associated to the second high-affinity site is observed [28,29], or that spin-labelled biT
I
I
I C
AKo~ 60
b
20
-20
-6(] 360
400
440
480 520 Wavelength (nm)
Fig. 10. Observed C D absorption for the system spin-labelled b i l i r u b i n / h u m a n serum albumin ( 2 . 5 . 1 0 - S M ) / b i l i r u b i n in 0.1 M phosphate buffer at pH 7.4; spin-labelled bilirubin/hum a n serum albumin = 0.7; b i l i r u b i n / h u m a n serum albumin = 0.0; 0.05; 0.1; 0.3; 0.5; 0.7; 1.0. Optical path length = 1 cm; gain = 5.10 -6.
Wavelength (nm)
Fi 8. i l. C D absorption assigned for the system bflirubin/hum a n serum albumin (curve c ) o b t a i n e d by subtracting curve b (spin-labelled b i l l r u b i n / h u m a n serum a l b u m i n = 0 . 8 ) from curve a (spin-labelled b i l i r u b i n / h u m a n serum aibumin/bilirubin = 0 : 8 / I / 0 : 5 ) ; curve d represents the C D curve for the binary system b i l i r u b i n / h u m a n serum albumin = 0.5.
350 lirubin is in part displaced by bilirubin from the first high-affinity site and enters the second site. Thus the contribution of the Type I curve, ascribed to bilirubin only, according to the preceding interpretation, is increased, and that of the Type II curve due to spin-labelled bilirubin at the first site is reduced. Some comments on the nature of the binding of spin-labelled bilirubin to human serum albumin can be drawn. Firstly, the interaction occurs through the outer end of spin-labelled bilirubin not including the aliphatic chain, which probably remains outside the macromolecules, as evidenced by ESR spectra. This behaviour prevails in both human serum albumin and bovine serum albumin interactions. In other words, there are affinity sites in human serum albumin and bovine serum albumin inducing CD of the same sign, although one cannot assert that the sites are the same. Moreover, sites of lower affinity are present, and in this case CD with an opposite sign is detected, according to whether the binding occurs with human serum albumin or bovine serum albumin. Assuming that the CD sign may be associated with the way the micromolecule enters the macromolecule, the CD curves related to spin-labelled bilirubin/human serum albumin and spin-labelled bilirubin/bovine serum albumin, holding the same sign, suggest that spin-labelled bilirubin occupies the highest-affinity binding sites of human serum albumin and bovine serum albumin and enters in the same way. Conclusions
Spin-labelled bilirubin gives rise to three types of association with human serum albumin (or bovine serum albumin). Two correspond to binding constants of about 107 and 106 1. mol-1, respectively, and the third one corresponds to a binding of about 103 1. mol-1. The latter association, involving the spin-labelled arm, was evidenced by ESR spectroscopy. The two former associations come out clearly on titration of the albumin by fluorescence quenching and leave the main part of the spin-labelled aliphatic chain so free that its environment is the same as that of spin-labelled bilirubin dissolved in aqueous solution. Moreover, it appears from the difference CD
spectra that binding with the site of highest-affinity induces a double Cotton effect, positive at lower wavelengths and negative at higher wavelengths. In contrast, the site of the lowest affinity is associated with a nearly symmetrical Cotton effect with an opposite sign. The analogy between the two high-affinity associations of spin-labelled bilirubin and bilirubin with human serum albumin appears to be very pronounced. For example, the magnitudes of the binding constant measured by fluorescence quenching are very similar, and in both cases a CD bi-signed Cotton effect results. It is likely that the protein site involved in the association with spin-labelled bilirubin and bilirubin are roughly the same, minor conformational changes excepted. On the other hand this finding is understandable by considering the close structural analogies between bilirubin and spin-labelled bilirubin, in which the bilirubin skeleton is left untouched, and a steric change of the side chain at the C-18 position occurs. This slight conformational difference of the chain at the distal position of the tetrapyrrolic moiety can lead to inversion of the Cotton effect (related to the high-affinity site) observed in the interaction with human serum albumin (but not with bovine serum albumin) by going from bilirubin to spin-labelled bilirubin. This is probably due to a reciprocal fitting of the conformations of the ligand and the protein which, in the thermodynamically favoured location, involves two conformations of opposite chirality for bilirubin and spin-labelled bilirubin. Conformational changes of the protein reflecting on the induced chirality of the ligand are known in the literature [30], and in particular for the bilirubin/ human serum albumin system, the bi-signed Cotton effect proved to be inverted by going from neutral to acid pH [12,28]. If, as it seems, spinlabelled bilirubin enters the sites of the albumin like the molecule of the natural pigment, the following conclusion may be drawn from the measurements reported on the spin-labelled bilirubin/ human serum albumin and bilirubin/human serum albumin complexes. In the interaction between bilirubin and human serum albumin the side chain C-18 is not directly involved in the binding, and this finding suggests that bilirubin enters the affinity sites of human serum albumin through deep insertion of only one of the two
351
extreme tings (or one pyrromethenone moiety), leaving the ring (or half molecule) free to originate a molecular conformation compatible both with its actual steric hindrance (different in bilirubin and spin-labelled bilirubin) and with the conformation preferred by the site, which is different for human serum albumin and bovine serum albumin and depends on pH. This is consistent with the changes of the Cotton effect sign, and could occur with the conformation of a tetrapyrrolic molecule either of the ridge-tile type (as in solid bilirubin or CHL13 solution [31,17]) or the helix type [32]. References 1 Brown, J.R. (1977) in Albumin Structure, Function and Uses (Rosender, V.M., Oratz, M., and Rothschild, M.A., eds.), pp. 27-52, Pergamon Press, New York 2 McMenamy, R.H. (1977) in Albumin Structure, Function and Uses (Rosender, V.M., Oratz, M. and Rothschild, M.A., eds.), pp. 143-158, Pergamon Press, New York 3 Arias, I.M. and Jansen, P. (1975) in Jaundice (Goresky, C.A. and Fischer, M.M., eds.), pp. 175-188, Plenum Press, New York 4 Schmid, R. (1975) Bilirubin: in Jaundice (Goresky, C.A. and Fischer, M.M., eds.), pp. 43-55, Plenum Press, New York 5 Scheidt, P.C., Mellitz, E.D., Hardy, J.B., Drage, J.S. and Boggs, T.R. (1977) J. Pediatr. 91,292-297 6 McDonagh, A.F. (1979) in The Porphyrins (Dolphin, D., eds.), pp. 294-491, Academic Press, New York 7 Brodersen, R. (1979) CRC Crit. Rev. Chem. Lab. Sci. 11, 305-399 8 Hsia, J.C., Kwan, N.N., Er, S.S., Wood, D.J. and Chance, G.W. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1542-1545 9 Wood, D.J. and Hsia, J.C. (1977) Biochem. Biophys. Res. Commun. 76, 863-868 10 Manitto, P. and Monti, D. (1973) Experientia 29, 137-138 11 Onishi, S., Kawade, N., Iton, S., Isobe, K. and Sugiyama, S. (1980) Biochem. J. 190, 527-532 12 Blauer, G., Harmatz, D. and Snir, J. (1972) Biochim. Biophys. Acta 278, 68-88
13 Dawson, R.M.C., Elliot, D.C., Elliot, W.H. and Jones, K.M. (1969) p. 490, Data for Biochemical Research, Clarendon Press, Oxford 14 Hampton, A., Slotin, L.A. and Chawla, R.R., (1976) J. Med. Chem. 19, 1279 15 McConnel, H.M., Deal, W. and Ogata, R.T. (1969) Biochemistry 8, 2580-2585 16 Levine, R.L. (1977) Clin. Chem. 23, 2292-2301 17 Manitto, P. and Monti, D. (1976) J.C.S. Chem. Commun. 122-123 18 Mugnoli, A., Manitto, P. and Monti, D. (1978) Nature 273, 568-569 19 Berde, C.B., Mudson, B.S., Simoni, R.D. and Skar, L.A. (1979) J. Biol. Chem. 254, 391-400 20 Klotz, I.M. and Hunston, D.L. (1971) Biochemistry 10, 3065-3069 21 Barzaghi, M.B., Gamba, A., Morosi, G. and Simonetta, M. (1978) J. Phys. Chem. 82, 2105-2114 22 Nordio, P.L. (1975) in Spin-Labeling, Theory and Applications (Berliner, L., ed.), pp. 5-52, Academic Press, New York 23 Freed, J.H. (1976) in Spin-Labeling, Theory and Applications (Berliner, L., ed.), pp. 53-132, Academic Press, New York 24 Reddoch, A.M. and Konishi, S. (1979) J. Chem. Phys. 70, 2121-2130 25 Chen, R.F. (1973) in Fluorescence techniques in cell biology (Thaen, A.A. and Serntz, M., eds.), pp. 273-282, SpringerVerlag, Berhn 26 Holzwarth, A.R., Langer, E., Lehner, H. and Scbaffner, K. (1980) Photochem. Photobiol. 32, 17-26 27 Witt, T.K. (1968) Bile Pigments, p. 270, Academic Press, New York 28 Kamisaka, K., Listowsky, I., Betheil, J.J. and Arias, I.M. (1974) Biochim. Biophys. Acta 365, 169-180 29 Beaven, G.H., D'Albis, A. and Gratze, W.B. (1973) Eur. J. Biochem. 33, 500-510 30 Branca, M. and Pispisa, B. (1977) J. Chem. Soc. Faraday I, 73, 213-229 31 Bonnet, R., Davis, J.E. and Hursthouse, M.B. (1976) Nature (London) 262, 326-328 32 Blauer, G. and Wagni~re, G. (1975) J. Am. Chem. Soc. 97, 1949-1954