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Optical properties of bilirubin-serum albumin complexes in aqueous solution. A comparison among albumins from different species
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
170,
375-383
(1975)
Optical Properties of Bilirubin-Serum Albumin Complexes in Aqueous Solution. A Comparison among Albumins from Different Species D. HARMATZ Department
of Biological
Chemistry, Received
AND
G. BLAUER
The Hebrew January
University
of Jerusalem,
(Israel)
2, 1975
Circular dichroism (CD) and light absorption were measured in the range of 300-550 nm on aqueous solutions (25-27°C) of complexes of bilirubin (2.5 x lo-” M) with charcoaltreated serum albumin [(5.0-12.5) x 10m5 M] from different species. Complexes ofhuman, bovine, goat, rabbit, porcine, and chicken albumins were compared at different pH values in the presence of 0.1 M NaCl. The observed CD spectra were found to depend largely on the pH of a given complex and also on the type of albumin at various constant pH values. Coupling between electric transition dipole moments within the dissymmetritally bound bilirubin molecule may contribute significantly to the observed optical activity in most cases, while other possible mechanisms have also been considered. The very large differences in the CD spectra observed in the present systems enable easy differentiation among the albumins from various species. Variations in the structure of the binding sites on the various albumins may account for at least part of the differences in the observed CD spectra of the respective bilirubin complexes.
CD’ measurements have recently been reported on the physiologically important complex bilirubin-HSA in aqueous solution (l-5). In our previous work (2, 3) (which also included some measurements on the bilirubin-BSA complex) the data obtained were analyzed on the basis of a proposed model which involved to a significant part electric transition dipole coupling within the bound and flexible bilirubin molecule. Other possible contributions to the large optical activity observed were also considered; these included the coupling of bilirubin transition moments with those of the protein and inherent dissymmetry formed in the chromophores of the bound bile pigment. In the absence of the protein, under otherwise similar conditions, free bilirubin in solution did not show measurable optical activity. CD and
light-absorption data on bilirubin complexes with serum albumins from goat and rabbit were also reported (6). It became apparent that CD is a very sensitive method for differentiating among the bilirubin complexes involving albumins from various mammalian species. The variations observed under comparable conditions were considered to reflect subtle differences at the respective binding sites of the albumins (6). The present report includes an extension of the comparison at different pH values among the complexes formed with human, bovine, goat, and rabbit albumins, as well as optical data on porcine and chicken albumins. It appears that the CD spectra (300450 nm) differ significantly for all species, as measured under comparable conditions. In several cases, the observed spectra are more complicated than those obtained previously for complexes involving HSA or BSA, and resolution, as well as interpretation of spectra, presents considerable difficulties.
’ Abbreviations: CD, circular dichroism; HSA, human serum albumin; BSA, bovine serum albumin; GSA, goat serum albumin; RSA, rabbit serum albumin; PSA, porcine serum albumin; CSA, chicken serum albumin. 375 Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
376
HARMATZ MATERIALS
AND
METHODS2
Bilirubin. Nutritional Biochemicals3 “certified” bilirubin was used (E ,,,Y = 60 mM-‘cm-’ at 453 nm in chloroform at 25°C). In order to test for the possible presence of isomers of bilirubin, the above sample was subjected to thin-layer chromatography (7). The main fraction, which was considered to constitute bilirubin IXa, was about 82% of the total substance. A sample of bilirubin from Sigma, fractionated by the same method, showed only trace amounts of secondary fractions. Complexes of bilirubin samples from each commercial source with excess HSA (pH 7.3) or BSA (pH 5.0), showed practically the same CD spectra in the visible region. Also, complexes prepared from alkaline bilirubin (N.B.C.) solutions, which were kept for various periods of time (up to 6 h) before complex formation with serum albumins from different species, did not indicate, within these time intervals, significant differences in the recorded CD spectra at both pH 7.4 and 4.0. It has been claimed (8) that significant isomerization of bilirubin proceeds, within the period of time considered, at the alkaline pH values (near pH 11) of the bilirubin stock solutions (8). It may be concluded from the above experiments, that if any amount of bilirubin isomers other that IXa is present in the systems investigated in this and previous work (see refs. 6, 2, and refs. cited therein), it should not significantly affect the CD spectra measured. Human serum albumin. Four times crystallized HSA was obtained from N.B.C. (for details, see ref.
2). Bovine serum albumin. Crystallized and lyophilized BSA was a preparation from Sigma (for details, see ref. 2). Goat serum albumin. GSA, Fraction V, powder, was purchased from N.B.C. (E,,,~ = 45 ? 1 m&cm-’ at 278-279 nm). This sample was used in most of the experiments given. A similar sample from Sigma was employed in several comparative measurements. Rabbit serum albumin, A crystallized RSA preparation from Pentex was used in most of the experiments; a crystallized sample from Schwarz-Mann was used for comparison. Both samples showed E,,,~ = 45 t 1 rnM-‘cm-’ at 278-279 nm. Porcine serum albumin. PSA, Fraction V, powder, was obtained from Pentex. For comparison, a sample of PSA from Schwarz-Mann (95% pure) was used. Both samples showed c,,,n = 47 * 2 mM-‘cm-1 at 278-279 nm. Chicken Serum albumin. CSA, Fraction V, was purchased from Pentex. For comparison, a sample * See ref. (2). 3 Abbreviated
N.B.C.
AND
BLAUER
from Sigma, Fraction V, was used. Both samples showed c,,,n = 35 k 3 mM-l.cm-’ at 278-279 nm. All serum albumins used in this work were treated with charcoal (9). The absence of fatty acids after this treatment was verified in several cases (determined as quoted in ref. 9). Samples of charcoal-treated albumins from all abovementioned animals were investigated in the analytical ultracentrifuge (Beckman, model El. In all cases, the apparent s~,,.~ values (concentration, 3.5 mg/ml) were found to be in the range of 4.2-4.4 S at neutral pH. As judged by the Schlieren curves, each sample appeared to contain about 90% (by weight) of albumin monomer (see ref. 3). Concentrations of the proteins are based on the Biuret method and on assumed molecular weights of 70,000 (see ref. 10). Millimolar extinction coefficients (c,ul given for the albumins, refer to neutral pH and 25°C. Other experimental details, including instrumentation and preparation of solutions, are as described previously (2). In the present work, the upper limit of the absorbance in CD measurements in the visible region was about 1.6. Electrolytes (including buffer) were always added to the protein solution before the bilirubin. Reference solutions for the complexes, in both light-absorption and CD measurements, contained the protein under similar conditions. Both molar extinction coefficients l and molar ellipticities [6] are based on the total bilirubin concentration. RESULTS
AND
DISCUSSION
(a) Reproducibility CD and light-absorption data obtained at both pH 7.4 and 4.0 for bilirubin complexes with albumin from various biological sources are summarized in Table I. In each case, albumins from two different commercial sources are compared. Part of the CD and light-absorption data show reasonable agreement with regard to the different sources. However, there are also large variations in some cases, particularly at pH 4.0. The largest differences observed are between the two commercial samples of GSA. Moreover, the sample from N.B.C. showed large, time-dependent changes at pH 4.0. Excluding the latter case, it appears that the differences among the bilirubin complexes involving albumins from different species at constant pH are much larger than the variations observed between the commercial preparations of a given albumin.
BILIRUBIN-SERUM
ALBUMIN TABLE
CD
AND LIGHT-ABSORPTION WITH ALBUMINS FROM Serum
albumin
Biological eource
Commercial eonrce
GSA
NBC Sigma
RSA
377
SPECIES
I
DATA (300-600 nm) IN AQUEOW SOLUTION OF COMPLEXES VARIOUS SPECIES, OBTAINED FROM DIFFERENT COMMERCIAL Observed CD-band extrema. Positions (nm) and corresponding molar ellipiticities x lo-’ in parentheses (degcm’.decimole-3 based on total bilirubin
PN Concn x 105 CM) 4.0
501(-9.2);
457c3.8); 465(-44);
Observed light-absorption maxima. Positions (nm) and E,,,M in parentheses (mu-‘.cm-‘1 based on total bilirobin
422(-0.8); 41X29.5)
395(0.5)*
448C35.5)" 456C49.4)’
35X0.6) 355U.O)
452C35.0)' 45OC33.6)
12.5
4.0
486C2.0); 484C4.1);
424.-2.9); 42X-5.5);
SM’
OF BILIRUBIN SOURCES’
PSA
Pentex SM
12.5
4.0
462.5(-91); 461(-82);
411(53) 411m
456C57.7) 45X54.4)
CSA
Pentex Sigma
12.5
4.0
478(-2.7); 488(-1.0);
417C2.2) 43X1.4)
443(41.4) 443C36.9)'
GSA
NBC Sigma
5.0
1.4
478(-17); 479(-12);
43OC6.3); 42X5.2);
RSA
Pentex SM
5.0
7.4
PSA
Pentex SM
5.0 12.5 5.0
7.4
CSA
Pentex Sigma
5.0 12.5 5.0
7.4
470(16); 469(12); LsOO(O.9);
41X-13); 412(-U);
456(-9.8); 459(-15) 459(-12)
-440(-9.5);
477.5(-62); 475(-81); 477(-73);
420(50) 41X65) 420(55)
387(-2.3) 387(-2.4)
458C63.5)' 46U53.8)
-34X1.2) -340(0.3)
425(42.8); 426t39.1);
-33W.l) -35OCO.6) -33X1.0)
463C55.4)' 462C56.0) 464C59.6)’
458(43.5F' 458C40.7)'
437C44.9) 434C48.0) 436C52.8)
a Bilirubin, 2.5 x 10e5 M; temp (CD), 27.0 + 0.5”C; temp (light absorption), 25 k 1°C; NaCI, 0.1 M; buffer Tris-HCI, 0.02 M, added at pH 7.4 only. CD measurements were started 20-30 min after preparation of the complex, unless otherwise indicated. In all cases, the ellipticity values given remained constant for an additional hour. Light-absorption spectra were recorded within about 1 h from the time of preparation of the complex. b Large changes of spectrum with time; final values given were reached after 200 min. ’ Average of two experiments. d Average of three experiments. ’ Abbreviation: SM. Schwarz-Mann.
Additional comparative data, not included in Table I (RSA samples at both pH 5 and 10, GSA at pH 5 and CSA at pH lo), also indicated similar CD spectra of the bilirubin complexes with albumins from different commercial sources (see Materials). (b) Effect of Albumin
Concentration
For all albumins, a fivefold molar excess of albumin over bilirubin (2.5 x 1O-5 M)
has been shown to be sufficient at pH 4.0 for the complete formation of CD spectra which do not change further at higher protein concentrations (see also ref. 31. Similarly, a twofold molar excess is sufficient at pH 7.4 for complexes involving either GSA or RSA; and also for HSA and BSA (see also ref. 3). However, for complexes with either PSA or CSA, a fivefold excess has been found to be required even at neutral pH (cf. ref. 111, in order to obtain CD
378
HARMATE
300
350
AND
BLAUER
450
400
500
550
hlnml
FIG. 1. CD spectra of complexes of bilirubin with serum albumins from different mammalian species, measured at pH 4.8. Bilirubin, 2.5 x lo-’ M; serum albumin, 1.2 x 10m4 M; NaCl, 0.1 M; temp 27.0 k 0.5”C. Measurements were started 20 min after adiustment of final pH, except for the RSA complex (45 min). Note two different scales for the ellipticities.
spectra which remain invariant at higher albumin concentrations. This requirement may be due either to relatively small association constants for the bilirubin complexes with either PSA or CSA, or to the possible presence, in each of these albumins, of a specific protein fraction which binds bilirubin selectively. 4 CD titrations5 of RSA, PSA, GSA, and CSA with bilirubin at pH 7.4 yielded apparent association constants of the order lo6 M-’ in all cases for the first specific site. In the case of RSA, a set of two binding sites was indicated. It is likely that a 1:l molar ratio is present in the complex at an excess of each of the albumins [see also HSA (5, 12) and BSA (1311. (c) Comparison of Bilirubin Complexes of HSA, BSA, GSA, and RSA, at Constant pH Values In a previous publication (6) spectra of bilirubin complexes with the above four different serum albumins were compared at both pH 7.4 and 4.0. Differences in sign of the CD bands and large variations in their magnitude have been observed among the various complexes, particularly at pH 4.0. Figures 1 and 2 add comparative data for the same albumin complexes at two additional pH values (4.8 and 10). All these pH values were chosen because, on ‘No direct evidence for such obtained in these cases. 5 Carried out by Mr. E. Lavie.
binding
could
be
lowering the pH of the HSA complex from values close to physiological conditions, a sharp transition in the CD spectra was observed near pH 5 and maximum values of the transformed spectrum were reached near pH 4 (ref. 2). At least in the case of the bilirubin-BSA complex it was shown that a molar ratio of 15 is sufficient at pH 5 for practically complete complex formation (13). At pH 4.8 (Fig. 11, the shape of the observed CD bands is similar in all cases, except for the RSA complex, which shows an additional, small positive band centered at about 500 nm. However, both GSA and BSA complexes show ellipticity values larger by one order of magnitude than those of the other complexes involving RSA and HSA. The corresponding light-absorption spectra, on the other hand, are similar for HSA and GSA complexes (Fig. 3). Figure 2 shows a comparison of CD data at pH 10. At this pH, HSA and RSA complexes are closer to each other than complexes involving BSA and GSA, which show various multiple bands of different sign. The corresponding light-absorption spectra are presented in Fig. 4. At pH 10, no measurements have been made at higher concentrations of serum albumin. (d> pH Dependence of the Various Complexes The dependence of both CD and lightabsorption spectra on pH, determined at
BILIRUBIN-SERUM
ALBUMIN
379
SPECIES
-20 -25 300
--50 I 350
... /'
I 400
450
- -60 I 500
I 550
600
X(nm) FIG. 2. CD spectra of complexes of bilirubin with serum albumins from different mammalian species, measured at pH 9.8 + 0.2. Bilirubin, 2.5 x lo-’ M; serum albumin, 5.0 x lo-’ M; NaCl, 0.1 M; temp 27.0 2 0.5”C. Time intervals between adjustment to final pH and measurement of complexes were as follows: BSA and RSA, 20 min; HSA, 80 min; GSA, 145 min. In the latter two cases, ellipticity values increased with time to the constant spectra shown.
FIG. 3. Light-absorption spectra of the systems given for Fig. 1 at pH 4.8. Temperature, 25 k 1°C. Measurements were started from 30 to 55 min after finai pH adjustment, except for the RSA complex (180 min).
several pH values in the range pH 4-10, can now be followed for each of the bilirubin complexes with a given albumin by combining the results of previous (2, 3, 6) and present measurements. In the case of HSA, there are always two proximate bands of opposite sign, both of which are inverted and shifted near pH 5. The absorption spectra do not differ much. BSA complexes also show two proximate bands of opposite sign. However, these are not in-
verted in their sign between pH 4-7.4, and reach maximum ellipticity values near pH 5 (Fig. 1). At pH 10, the ellipticities diminish and new smaller bands appear (Fig. 2). Light-absorption spectra at different pH values have been treated previously (13, 14). Bilirubin-GSA complexes resemble corresponding BSA complexes at both pH 4 and 4.8, with the exception of a sample from N.B.C. at pH 4 (see above), which showed a complicated kinetic behaviour and multibanded CD spectra (6). At pH 7.4, the main bands apparently retain their sign and are shifted to longer wavelengths, with an additional negative band centered near 400 nm (6). In this region, small positive ellipticities are recorded at pH 10 (Fig. 2). The corresponding lightabsorption spectra show gradual shifts to longer wavelengths and an increase in the observed band maxima, as the pH increases. Freshly drawn bovine or goat blood sera from single animals also showed optical activity at both pH 7.4 and 4.0. However, agreement with the CD-band positions of the respective bilirubin-albumin complexes was fair at pH 4 only. Complexes of bilirubin with RSA differ at pH 4.0 from the three other albumin
380
FIG. 4. Light-absorption Measurements were started
HARMATZ
AND
BLAUER
spectra of the systems given for Fig. 2 at pH 9.8 + 0.2. Temperature from 45 to 60 min after the final pH adjustment.
complexes described above, as they show a small, but positive CD band at the longwavelength end, in addition to several other bands of opposite sign (6). At pH 4.8 (Fig. l), the small band near 500 nm persists, while the negative ellipticity near 420 nm observed at pH 4.0 becomes positive at pH 4.8 and negative again at pH 7.4 (ref. 6) and at pH 10 (Fig. 2). At these latter two pH values, the CD spectra of the RSA complex are similar to those of the HSA complex. The light-absorption spectra of the corresponding RSA complexes show lower band maxima at most pH values. At pH 7.4, two distinct and proximate maxima are observed (6), in contrast to the complexes of the other species. At pH 4.0, the light-absorption bands of the complexes of all four species appear to be split (6). The observed absorption bands of either HSA or BSA complexes with bilirubin in the visible region could be resolved fairly well into two Gaussian curves at various pH values (2). NO correlation of the CD spectral changes with the N + F transition of albumin (midpoint near pH 4) is apparent in the systems described above.
(e) Complexes
of Bilirubin PSA or CSA
25 ? 1°C.
with
either
CD and light-absorption spectra of the corresponding bilirubin complexes are given at two pH values only (pH 7.4 and 4.0; Figs. 5-7). At pH 4.0, two proximate CD bands of opposite sign are again observed. However, the bands are much larger in the case of the PSA complex (Fig. 5). The spectrum of the latter is similar to that of the corresponding HSA complex at pH 4, although the band extrema are slightly shifted. At pH 7.4 (Fig. 6), the CSA complex shows much larger ellipticity values than the PSA complex. However, while the CD curves of the CSA complex appear to retain their general shape with respect to pH 4, the PSA complex shows relatively small and positive CD bands near 500 and 330 nm, respectively; the positive band near 410 nm is not apparent and additional negative CD bands appear near 450 nm. This latter PSA-bilirubin spectrum obtained at pH 7.4 is unique and is not similar to any spectrum described above. Comparing the light-absorption spectra
BILIRLJBIN-SERUM
20 -
:: :a, -i .A-5:. . L -iu--0 0 2” h
0 -.-.-__
,‘,.-
ALBUMIN
’
./’
,. -y,
c h,Ck”
-.--4--*-
‘\
‘\
*
A’, lbrcinc ,
-80 350
400
, T,; c .I 9’
,I’ \
-60 -
-100 300
‘\
\ >
~20 -
\
/
\
F> : - 0 - - -z bays.. -~5 ,+ --2 - ~10 -;
-5
‘!.; $
~40 -
381
SPECIES
\
\. ,
f
’
I 1 ’
7-
- 15 - ~20
450
500
-25 550
hlnml
Frc. 5. CD spectra of complexes of bilirubin with PSA and CSA at pH 4.0. Bilirubin, albumin, 1.2 x lo-” M; NaCl, 0.1 M; temp 27.9 + 0.5”C. Measurements were started adjustment of the final pH. Note two different scales for the ellipticities.
Porc,ne \
0 -60
\
-80 -100 300
‘\ ,’ L,’
\ \
350
/’
450
---
1 --15
Q
-20
\ ._ ,-_’
400
2.5 x 10e5 M; serum about 30 min after
I 500
-25 550
AlnmJ
6. CD spectra of complexes of bilirubin with PSA and CSA at pH 7.4. Bilirubin, 2.5 albumin, 5.0 x 10m5 M; NaCl, 0.1 M; ‘Iris-HCl buffer, 0.02 M; temp 27.0 + 0.5%. Measurements 20 min after adjustment of the final pH. Note two different scales for the ellipticities. FIG.
of the above complexes (Fig. 71, higher band maxima are observed for the PSA complexes at both pH 4.0 and 7.4. It should again be noted (see above, and Table I) that at pH 7.4, both PSA and GSA were not in sufficient excess over bilirubin to give the maximum ellipticity and absorption values. (fl Interpretation and Significance Results Obtained
of the
CD and light-absorption spectra of HSA and BSA complexes with bilirubin have been analyzed and interpreted (2, 27). A substantial part of the optical activity observed has been attributed to coupling between electric transition dipole moments of the dipyrromethene chromophores of dis-
x
10e5 M; serum were started
symmetrically bound bilirubin. Changes in the sign and large variations in the magnitude of the CD bands observed under various conditions have been correlated with changes in the relative position of these transition dipoles, either within a single flexible bilirubin molecule or between neighboring bilirubin molecules. Other contributions to the optical activity, such as the coupling of bilirubin transition dipoles with those of protein (aromatic) groups (151, or the formation of skewed chromophores upon binding of the bilirubin molecule (see ref. 16 for urobilins, and ref. 17 for biliverdin), should also be considered. A previous comparison of visible-range CD spectra of complexes of bilirubin with
HARMA’IZ
382
350
400
450
500
550
X(nm)
FIG. 7. Light-absorption spectra of the systems given for Figs. 5 and 6. Temperature 25 f 1°C. Measurements were started from 40 to 60 min after the final pH adjustment.
serum albumins from different sources or species has been considered to constitute a very sensitive measure of differences at the respective binding sites (2, 6). Conformational differences among the albumins and/or differences in the electrochemical properties of binding groups have been assumed to account for the effects observed (2, 6). It may be noted that only for complexes involving either HSA or RSA is an inversion with pH in the sign of the main observed CD bands apparent. It is therefore unlikely that this inversion is due mainly to changes in the orientation of the transition dipole moments of bilirubin caused by ionization of chromophoric groups of the bile pigment. More likely, changes in the chirality of the bound bilirubin molecule are involved (2, 27). The present extended comparison of complexes involving serum albumins from a larger number of species confirms the high sensitivity of CD spectra as an analytical tool for differentiating among the various albumins. This sensitivity is apparently much greater in CD spectra than in corresponding light-absorption spectra (M-20) or in CD data of complexes involving various albumins and drugs or dyes (21, 22), instead of bilirubin. While there appear to be no large differences in sedimentation veloc-
AND BLAUER
ity (see above) or, most likely, also in molecular weight (10, 23) among the albumins from different vertebrate species, some differences in viscosity or titration curves have been observed between HSA and BSA (24). Also, the fluorescence properties of relaxation processesof bound bilirubin were found to vary for complexes with albumins from different species (28). Despite general similarities in amino acid composition (29, the large variations observed in the CD spectra of the various bilirubin-albumin complexes may reflect certain differences in the primary structure of the various albumins (23), which in turn could affect the tertiary structure and hence the nature of the binding sites of the various albumins. As already mentioned, possible heterogeneity in the various albumins (see, for example, ref. 26) may account, in some cases, for the differences in binding observed, since a specific fraction may bind bilirubin selectively. In the previously investigated systems involving HSA and BSA complexes at various pH values, the main CD bands near 460-470 nm and 405-425 nm, respectively, could be resolved fairly well into two Gaussian curves of opposite sign. However, the rotatory strengths of these two bands were not quite equal in many cases, indicating mixing with other states or additional contributions to the optical activity (2). Many of the visible-range CD bands given in the present and previous work (6) for various bilirubin-albumin complexes show a more complicated pattern, e.g., complexes of GSA at pH 7.4 and 4.0, of RSA at pH 4.0 (ref. 6, Figs. 1 and 2) or of PSA at pH 7.4 (Fig. 6). It seems possible that equilibria between two or more types of complexes are observed in some cases. For the GSA and RSA complexes at pH 7.4, resolution of the experimental CD and light-absorption spectra into Gaussian curves has now been carried out. In the case of the bilirubin-RSA complex, at least three Gaussians are required in the range 300-550 nm, with fair agreement in position and band width of the resolved CD and lightabsorption bands. If exciton bands of equal and opposite rotatory strength are taken into account, an additional positive Gaus-
BILIRUBIN-SERUM
sian curve is required in the CD spectrum. In the case of the GSA complex at pH 7.4, at least four Gaussian curves are necessary for reasonable resolution in the same wavelength range. However, there is less agreement between the parameters of CD and light-absorption bands than in the previous case. A more detailed treatment of the resolution and interpretation of observed spectra is under investigation. ACKNOWLEDGMENTS The skillful technical assistance and of Mr. P. Yanai is appreciated. supported by a Grant (No. 111772) Volkswagenwerk.
of Mrs. J. Silfen This work was from the Stiftung
REFERENCES 1. BLAUER, G., HARMATZ, D., AND NAPARSTEK, A. (1970) FEBS Lett. 9, 53-55. 2. BLAUER, G., HARMATZ, D., AND SNIR, J. (1972) Biochim. Biophys. Acta 278, 68-88. 3. BLAUER, G. AND HARMATZ, D. (1972) Biochim. Biophys. Acta 278, 89-100. 4. WOOLLEY, P. V., III AND HUNTER, M. J. (1970) Arch. Biochem. Biophys. 40, 197-209. 5. BEAVEN, G. H., D’ALBIS, A., AND GRATZER, W. B. (1973) Eur. J. Biochem. 33, 500-509. 6. BLAUER, G. AND HARMATZ, D. (1973) Jerusalem Symposia on Quantum Chemistry and Biochemistry (Bergmann, E. D. and Pullman, B. eds.), Vol. 5, pp. 709-714, Isr. Acad. Sci. Human., Jerusalem. 7. MCDONAGH, A. F. AND ASSISI, F. (1971) FEBS Z&t. 18, 315-317. 8. MCDONAGH, A. F. AND ASSISI, F. (1972) Biochem. J. 129, 797-800. 9. CHEN, R. F. (1967) J. Biol. Chem. 242,173-181. 10. PHELPS, R. A. AND PUTNAM, F. W. (1960) The Plasma Proteins (Putnam, F. W., ed.), Vol. 1,
ALBUMIN
SPECIES
383
pp. 143-178, Academic Press, New York. 11. SCHMID, R., DIAMOND, I., HAMMAKER, L., AND GUNDERSEN, C. B. (1965) Nature (London) 206, 1041-1043. 12. JACOBSEN, J. (1969) FEBS Lett. 5, 112-114. 13. BLAUER, G. AND KING, T. E. (1970) J. Biol. Chem. 245, 372-381. 14. WENNBERG, R. P. AND COWGER, M. L. (1973) Clin. Chin. Acta 43, 55-64. 15. Hsu, M. C. AND WOODY, R. W. (1971) J. Amer. Chem. Sot. 93, 3515-3525. 16. MOSCOWITZ, A., KRUEGER, W. C., KAY, I. T., SKEWES, G., AND BRUCKENSTEIN, S. (1964) Proc. Nat. Acad. Sci. USA 52, 1190-1194. 17. BLAUER, G. AND ZVILICHOVSKY, B. (1973) Zsr. J. Chem. 11.435-443. 18. PEMBERTON, J. R. AND DE JONG, J. (1971)Anal. Biochem. 43, 575-581. 19. WITIAK, D. T. AND WHITEHOUSE, M. W. (1969) Biochem. Pharmacol. 18, 971-977. 20. BAXTER, J. H. (1964) Arch. Biochem. Biophys. 108,375-383. 21. CHIGNELL, C. F. (1969) Mol. Pharmacol. 5, 244252,455-462. 22. BRAND, J. G. AND TORIBARA, T. Y. (1973) Biochem. Biophys. Res. Commun. 52, 511516. 23. PUTNAM, F. W. (1965) The Proteins (Neurath, H., ed.), 2nd edn, Vol. 3, pp. 154-267, Academic Press, New York-London. 24. STEINHARDT, J., KRIJN, J., AND LEIDY, J. G. (1971) Biochemistry 10, 4005-4014. 25. FOSTER, J. F. (1967) Encyclopedia of Biochemistry (Williams, R. J. and Lansford, E. M., Jr., eds.), p. 20, Reinhold, New York. 26. WONG, Kc P. AND FOSTER, J. F. (1969) Biochemisty 8,4104-4108. 27. BLAUER, G. AND WAGNIPRE, G. (1975) J. Amer. Chem. Sot. 97, 1949-1954. 28. CHEN, R. F. (1974)Arch. Biochem. Biophys. 160, 106-112.
OF BIOCHEMISTRY
AND
BIOPHYSICS
170,
375-383
(1975)
Optical Properties of Bilirubin-Serum Albumin Complexes in Aqueous Solution. A Comparison among Albumins from Different Species D. HARMATZ Department
of Biological
Chemistry, Received
AND
G. BLAUER
The Hebrew January
University
of Jerusalem,
(Israel)
2, 1975
Circular dichroism (CD) and light absorption were measured in the range of 300-550 nm on aqueous solutions (25-27°C) of complexes of bilirubin (2.5 x lo-” M) with charcoaltreated serum albumin [(5.0-12.5) x 10m5 M] from different species. Complexes ofhuman, bovine, goat, rabbit, porcine, and chicken albumins were compared at different pH values in the presence of 0.1 M NaCl. The observed CD spectra were found to depend largely on the pH of a given complex and also on the type of albumin at various constant pH values. Coupling between electric transition dipole moments within the dissymmetritally bound bilirubin molecule may contribute significantly to the observed optical activity in most cases, while other possible mechanisms have also been considered. The very large differences in the CD spectra observed in the present systems enable easy differentiation among the albumins from various species. Variations in the structure of the binding sites on the various albumins may account for at least part of the differences in the observed CD spectra of the respective bilirubin complexes.
CD’ measurements have recently been reported on the physiologically important complex bilirubin-HSA in aqueous solution (l-5). In our previous work (2, 3) (which also included some measurements on the bilirubin-BSA complex) the data obtained were analyzed on the basis of a proposed model which involved to a significant part electric transition dipole coupling within the bound and flexible bilirubin molecule. Other possible contributions to the large optical activity observed were also considered; these included the coupling of bilirubin transition moments with those of the protein and inherent dissymmetry formed in the chromophores of the bound bile pigment. In the absence of the protein, under otherwise similar conditions, free bilirubin in solution did not show measurable optical activity. CD and
light-absorption data on bilirubin complexes with serum albumins from goat and rabbit were also reported (6). It became apparent that CD is a very sensitive method for differentiating among the bilirubin complexes involving albumins from various mammalian species. The variations observed under comparable conditions were considered to reflect subtle differences at the respective binding sites of the albumins (6). The present report includes an extension of the comparison at different pH values among the complexes formed with human, bovine, goat, and rabbit albumins, as well as optical data on porcine and chicken albumins. It appears that the CD spectra (300450 nm) differ significantly for all species, as measured under comparable conditions. In several cases, the observed spectra are more complicated than those obtained previously for complexes involving HSA or BSA, and resolution, as well as interpretation of spectra, presents considerable difficulties.
’ Abbreviations: CD, circular dichroism; HSA, human serum albumin; BSA, bovine serum albumin; GSA, goat serum albumin; RSA, rabbit serum albumin; PSA, porcine serum albumin; CSA, chicken serum albumin. 375 Copyright All rights
0 1975 by Academic Press, of reproduction in any form
Inc. reserved.
376
HARMATZ MATERIALS
AND
METHODS2
Bilirubin. Nutritional Biochemicals3 “certified” bilirubin was used (E ,,,Y = 60 mM-‘cm-’ at 453 nm in chloroform at 25°C). In order to test for the possible presence of isomers of bilirubin, the above sample was subjected to thin-layer chromatography (7). The main fraction, which was considered to constitute bilirubin IXa, was about 82% of the total substance. A sample of bilirubin from Sigma, fractionated by the same method, showed only trace amounts of secondary fractions. Complexes of bilirubin samples from each commercial source with excess HSA (pH 7.3) or BSA (pH 5.0), showed practically the same CD spectra in the visible region. Also, complexes prepared from alkaline bilirubin (N.B.C.) solutions, which were kept for various periods of time (up to 6 h) before complex formation with serum albumins from different species, did not indicate, within these time intervals, significant differences in the recorded CD spectra at both pH 7.4 and 4.0. It has been claimed (8) that significant isomerization of bilirubin proceeds, within the period of time considered, at the alkaline pH values (near pH 11) of the bilirubin stock solutions (8). It may be concluded from the above experiments, that if any amount of bilirubin isomers other that IXa is present in the systems investigated in this and previous work (see refs. 6, 2, and refs. cited therein), it should not significantly affect the CD spectra measured. Human serum albumin. Four times crystallized HSA was obtained from N.B.C. (for details, see ref.
2). Bovine serum albumin. Crystallized and lyophilized BSA was a preparation from Sigma (for details, see ref. 2). Goat serum albumin. GSA, Fraction V, powder, was purchased from N.B.C. (E,,,~ = 45 ? 1 m&cm-’ at 278-279 nm). This sample was used in most of the experiments given. A similar sample from Sigma was employed in several comparative measurements. Rabbit serum albumin, A crystallized RSA preparation from Pentex was used in most of the experiments; a crystallized sample from Schwarz-Mann was used for comparison. Both samples showed E,,,~ = 45 t 1 rnM-‘cm-’ at 278-279 nm. Porcine serum albumin. PSA, Fraction V, powder, was obtained from Pentex. For comparison, a sample of PSA from Schwarz-Mann (95% pure) was used. Both samples showed c,,,n = 47 * 2 mM-‘cm-1 at 278-279 nm. Chicken Serum albumin. CSA, Fraction V, was purchased from Pentex. For comparison, a sample * See ref. (2). 3 Abbreviated
N.B.C.
AND
BLAUER
from Sigma, Fraction V, was used. Both samples showed c,,,n = 35 k 3 mM-l.cm-’ at 278-279 nm. All serum albumins used in this work were treated with charcoal (9). The absence of fatty acids after this treatment was verified in several cases (determined as quoted in ref. 9). Samples of charcoal-treated albumins from all abovementioned animals were investigated in the analytical ultracentrifuge (Beckman, model El. In all cases, the apparent s~,,.~ values (concentration, 3.5 mg/ml) were found to be in the range of 4.2-4.4 S at neutral pH. As judged by the Schlieren curves, each sample appeared to contain about 90% (by weight) of albumin monomer (see ref. 3). Concentrations of the proteins are based on the Biuret method and on assumed molecular weights of 70,000 (see ref. 10). Millimolar extinction coefficients (c,ul given for the albumins, refer to neutral pH and 25°C. Other experimental details, including instrumentation and preparation of solutions, are as described previously (2). In the present work, the upper limit of the absorbance in CD measurements in the visible region was about 1.6. Electrolytes (including buffer) were always added to the protein solution before the bilirubin. Reference solutions for the complexes, in both light-absorption and CD measurements, contained the protein under similar conditions. Both molar extinction coefficients l and molar ellipticities [6] are based on the total bilirubin concentration. RESULTS
AND
DISCUSSION
(a) Reproducibility CD and light-absorption data obtained at both pH 7.4 and 4.0 for bilirubin complexes with albumin from various biological sources are summarized in Table I. In each case, albumins from two different commercial sources are compared. Part of the CD and light-absorption data show reasonable agreement with regard to the different sources. However, there are also large variations in some cases, particularly at pH 4.0. The largest differences observed are between the two commercial samples of GSA. Moreover, the sample from N.B.C. showed large, time-dependent changes at pH 4.0. Excluding the latter case, it appears that the differences among the bilirubin complexes involving albumins from different species at constant pH are much larger than the variations observed between the commercial preparations of a given albumin.
BILIRUBIN-SERUM
ALBUMIN TABLE
CD
AND LIGHT-ABSORPTION WITH ALBUMINS FROM Serum
albumin
Biological eource
Commercial eonrce
GSA
NBC Sigma
RSA
377
SPECIES
I
DATA (300-600 nm) IN AQUEOW SOLUTION OF COMPLEXES VARIOUS SPECIES, OBTAINED FROM DIFFERENT COMMERCIAL Observed CD-band extrema. Positions (nm) and corresponding molar ellipiticities x lo-’ in parentheses (degcm’.decimole-3 based on total bilirubin
PN Concn x 105 CM) 4.0
501(-9.2);
457c3.8); 465(-44);
Observed light-absorption maxima. Positions (nm) and E,,,M in parentheses (mu-‘.cm-‘1 based on total bilirobin
422(-0.8); 41X29.5)
395(0.5)*
448C35.5)" 456C49.4)’
35X0.6) 355U.O)
452C35.0)' 45OC33.6)
12.5
4.0
486C2.0); 484C4.1);
424.-2.9); 42X-5.5);
SM’
OF BILIRUBIN SOURCES’
PSA
Pentex SM
12.5
4.0
462.5(-91); 461(-82);
411(53) 411m
456C57.7) 45X54.4)
CSA
Pentex Sigma
12.5
4.0
478(-2.7); 488(-1.0);
417C2.2) 43X1.4)
443(41.4) 443C36.9)'
GSA
NBC Sigma
5.0
1.4
478(-17); 479(-12);
43OC6.3); 42X5.2);
RSA
Pentex SM
5.0
7.4
PSA
Pentex SM
5.0 12.5 5.0
7.4
CSA
Pentex Sigma
5.0 12.5 5.0
7.4
470(16); 469(12); LsOO(O.9);
41X-13); 412(-U);
456(-9.8); 459(-15) 459(-12)
-440(-9.5);
477.5(-62); 475(-81); 477(-73);
420(50) 41X65) 420(55)
387(-2.3) 387(-2.4)
458C63.5)' 46U53.8)
-34X1.2) -340(0.3)
425(42.8); 426t39.1);
-33W.l) -35OCO.6) -33X1.0)
463C55.4)' 462C56.0) 464C59.6)’
458(43.5F' 458C40.7)'
437C44.9) 434C48.0) 436C52.8)
a Bilirubin, 2.5 x 10e5 M; temp (CD), 27.0 + 0.5”C; temp (light absorption), 25 k 1°C; NaCI, 0.1 M; buffer Tris-HCI, 0.02 M, added at pH 7.4 only. CD measurements were started 20-30 min after preparation of the complex, unless otherwise indicated. In all cases, the ellipticity values given remained constant for an additional hour. Light-absorption spectra were recorded within about 1 h from the time of preparation of the complex. b Large changes of spectrum with time; final values given were reached after 200 min. ’ Average of two experiments. d Average of three experiments. ’ Abbreviation: SM. Schwarz-Mann.
Additional comparative data, not included in Table I (RSA samples at both pH 5 and 10, GSA at pH 5 and CSA at pH lo), also indicated similar CD spectra of the bilirubin complexes with albumins from different commercial sources (see Materials). (b) Effect of Albumin
Concentration
For all albumins, a fivefold molar excess of albumin over bilirubin (2.5 x 1O-5 M)
has been shown to be sufficient at pH 4.0 for the complete formation of CD spectra which do not change further at higher protein concentrations (see also ref. 31. Similarly, a twofold molar excess is sufficient at pH 7.4 for complexes involving either GSA or RSA; and also for HSA and BSA (see also ref. 3). However, for complexes with either PSA or CSA, a fivefold excess has been found to be required even at neutral pH (cf. ref. 111, in order to obtain CD
378
HARMATE
300
350
AND
BLAUER
450
400
500
550
hlnml
FIG. 1. CD spectra of complexes of bilirubin with serum albumins from different mammalian species, measured at pH 4.8. Bilirubin, 2.5 x lo-’ M; serum albumin, 1.2 x 10m4 M; NaCl, 0.1 M; temp 27.0 k 0.5”C. Measurements were started 20 min after adiustment of final pH, except for the RSA complex (45 min). Note two different scales for the ellipticities.
spectra which remain invariant at higher albumin concentrations. This requirement may be due either to relatively small association constants for the bilirubin complexes with either PSA or CSA, or to the possible presence, in each of these albumins, of a specific protein fraction which binds bilirubin selectively. 4 CD titrations5 of RSA, PSA, GSA, and CSA with bilirubin at pH 7.4 yielded apparent association constants of the order lo6 M-’ in all cases for the first specific site. In the case of RSA, a set of two binding sites was indicated. It is likely that a 1:l molar ratio is present in the complex at an excess of each of the albumins [see also HSA (5, 12) and BSA (1311. (c) Comparison of Bilirubin Complexes of HSA, BSA, GSA, and RSA, at Constant pH Values In a previous publication (6) spectra of bilirubin complexes with the above four different serum albumins were compared at both pH 7.4 and 4.0. Differences in sign of the CD bands and large variations in their magnitude have been observed among the various complexes, particularly at pH 4.0. Figures 1 and 2 add comparative data for the same albumin complexes at two additional pH values (4.8 and 10). All these pH values were chosen because, on ‘No direct evidence for such obtained in these cases. 5 Carried out by Mr. E. Lavie.
binding
could
be
lowering the pH of the HSA complex from values close to physiological conditions, a sharp transition in the CD spectra was observed near pH 5 and maximum values of the transformed spectrum were reached near pH 4 (ref. 2). At least in the case of the bilirubin-BSA complex it was shown that a molar ratio of 15 is sufficient at pH 5 for practically complete complex formation (13). At pH 4.8 (Fig. 11, the shape of the observed CD bands is similar in all cases, except for the RSA complex, which shows an additional, small positive band centered at about 500 nm. However, both GSA and BSA complexes show ellipticity values larger by one order of magnitude than those of the other complexes involving RSA and HSA. The corresponding light-absorption spectra, on the other hand, are similar for HSA and GSA complexes (Fig. 3). Figure 2 shows a comparison of CD data at pH 10. At this pH, HSA and RSA complexes are closer to each other than complexes involving BSA and GSA, which show various multiple bands of different sign. The corresponding light-absorption spectra are presented in Fig. 4. At pH 10, no measurements have been made at higher concentrations of serum albumin. (d> pH Dependence of the Various Complexes The dependence of both CD and lightabsorption spectra on pH, determined at
BILIRUBIN-SERUM
ALBUMIN
379
SPECIES
-20 -25 300
--50 I 350
... /'
I 400
450
- -60 I 500
I 550
600
X(nm) FIG. 2. CD spectra of complexes of bilirubin with serum albumins from different mammalian species, measured at pH 9.8 + 0.2. Bilirubin, 2.5 x lo-’ M; serum albumin, 5.0 x lo-’ M; NaCl, 0.1 M; temp 27.0 2 0.5”C. Time intervals between adjustment to final pH and measurement of complexes were as follows: BSA and RSA, 20 min; HSA, 80 min; GSA, 145 min. In the latter two cases, ellipticity values increased with time to the constant spectra shown.
FIG. 3. Light-absorption spectra of the systems given for Fig. 1 at pH 4.8. Temperature, 25 k 1°C. Measurements were started from 30 to 55 min after finai pH adjustment, except for the RSA complex (180 min).
several pH values in the range pH 4-10, can now be followed for each of the bilirubin complexes with a given albumin by combining the results of previous (2, 3, 6) and present measurements. In the case of HSA, there are always two proximate bands of opposite sign, both of which are inverted and shifted near pH 5. The absorption spectra do not differ much. BSA complexes also show two proximate bands of opposite sign. However, these are not in-
verted in their sign between pH 4-7.4, and reach maximum ellipticity values near pH 5 (Fig. 1). At pH 10, the ellipticities diminish and new smaller bands appear (Fig. 2). Light-absorption spectra at different pH values have been treated previously (13, 14). Bilirubin-GSA complexes resemble corresponding BSA complexes at both pH 4 and 4.8, with the exception of a sample from N.B.C. at pH 4 (see above), which showed a complicated kinetic behaviour and multibanded CD spectra (6). At pH 7.4, the main bands apparently retain their sign and are shifted to longer wavelengths, with an additional negative band centered near 400 nm (6). In this region, small positive ellipticities are recorded at pH 10 (Fig. 2). The corresponding lightabsorption spectra show gradual shifts to longer wavelengths and an increase in the observed band maxima, as the pH increases. Freshly drawn bovine or goat blood sera from single animals also showed optical activity at both pH 7.4 and 4.0. However, agreement with the CD-band positions of the respective bilirubin-albumin complexes was fair at pH 4 only. Complexes of bilirubin with RSA differ at pH 4.0 from the three other albumin
380
FIG. 4. Light-absorption Measurements were started
HARMATZ
AND
BLAUER
spectra of the systems given for Fig. 2 at pH 9.8 + 0.2. Temperature from 45 to 60 min after the final pH adjustment.
complexes described above, as they show a small, but positive CD band at the longwavelength end, in addition to several other bands of opposite sign (6). At pH 4.8 (Fig. l), the small band near 500 nm persists, while the negative ellipticity near 420 nm observed at pH 4.0 becomes positive at pH 4.8 and negative again at pH 7.4 (ref. 6) and at pH 10 (Fig. 2). At these latter two pH values, the CD spectra of the RSA complex are similar to those of the HSA complex. The light-absorption spectra of the corresponding RSA complexes show lower band maxima at most pH values. At pH 7.4, two distinct and proximate maxima are observed (6), in contrast to the complexes of the other species. At pH 4.0, the light-absorption bands of the complexes of all four species appear to be split (6). The observed absorption bands of either HSA or BSA complexes with bilirubin in the visible region could be resolved fairly well into two Gaussian curves at various pH values (2). NO correlation of the CD spectral changes with the N + F transition of albumin (midpoint near pH 4) is apparent in the systems described above.
(e) Complexes
of Bilirubin PSA or CSA
25 ? 1°C.
with
either
CD and light-absorption spectra of the corresponding bilirubin complexes are given at two pH values only (pH 7.4 and 4.0; Figs. 5-7). At pH 4.0, two proximate CD bands of opposite sign are again observed. However, the bands are much larger in the case of the PSA complex (Fig. 5). The spectrum of the latter is similar to that of the corresponding HSA complex at pH 4, although the band extrema are slightly shifted. At pH 7.4 (Fig. 6), the CSA complex shows much larger ellipticity values than the PSA complex. However, while the CD curves of the CSA complex appear to retain their general shape with respect to pH 4, the PSA complex shows relatively small and positive CD bands near 500 and 330 nm, respectively; the positive band near 410 nm is not apparent and additional negative CD bands appear near 450 nm. This latter PSA-bilirubin spectrum obtained at pH 7.4 is unique and is not similar to any spectrum described above. Comparing the light-absorption spectra
BILIRLJBIN-SERUM
20 -
:: :a, -i .A-5:. . L -iu--0 0 2” h
0 -.-.-__
,‘,.-
ALBUMIN
’
./’
,. -y,
c h,Ck”
-.--4--*-
‘\
‘\
*
A’, lbrcinc ,
-80 350
400
, T,; c .I 9’
,I’ \
-60 -
-100 300
‘\
\ >
~20 -
\
/
\
F> : - 0 - - -z bays.. -~5 ,+ --2 - ~10 -;
-5
‘!.; $
~40 -
381
SPECIES
\
\. ,
f
’
I 1 ’
7-
- 15 - ~20
450
500
-25 550
hlnml
Frc. 5. CD spectra of complexes of bilirubin with PSA and CSA at pH 4.0. Bilirubin, albumin, 1.2 x lo-” M; NaCl, 0.1 M; temp 27.9 + 0.5”C. Measurements were started adjustment of the final pH. Note two different scales for the ellipticities.
Porc,ne \
0 -60
\
-80 -100 300
‘\ ,’ L,’
\ \
350
/’
450
---
1 --15
Q
-20
\ ._ ,-_’
400
2.5 x 10e5 M; serum about 30 min after
I 500
-25 550
AlnmJ
6. CD spectra of complexes of bilirubin with PSA and CSA at pH 7.4. Bilirubin, 2.5 albumin, 5.0 x 10m5 M; NaCl, 0.1 M; ‘Iris-HCl buffer, 0.02 M; temp 27.0 + 0.5%. Measurements 20 min after adjustment of the final pH. Note two different scales for the ellipticities. FIG.
of the above complexes (Fig. 71, higher band maxima are observed for the PSA complexes at both pH 4.0 and 7.4. It should again be noted (see above, and Table I) that at pH 7.4, both PSA and GSA were not in sufficient excess over bilirubin to give the maximum ellipticity and absorption values. (fl Interpretation and Significance Results Obtained
of the
CD and light-absorption spectra of HSA and BSA complexes with bilirubin have been analyzed and interpreted (2, 27). A substantial part of the optical activity observed has been attributed to coupling between electric transition dipole moments of the dipyrromethene chromophores of dis-
x
10e5 M; serum were started
symmetrically bound bilirubin. Changes in the sign and large variations in the magnitude of the CD bands observed under various conditions have been correlated with changes in the relative position of these transition dipoles, either within a single flexible bilirubin molecule or between neighboring bilirubin molecules. Other contributions to the optical activity, such as the coupling of bilirubin transition dipoles with those of protein (aromatic) groups (151, or the formation of skewed chromophores upon binding of the bilirubin molecule (see ref. 16 for urobilins, and ref. 17 for biliverdin), should also be considered. A previous comparison of visible-range CD spectra of complexes of bilirubin with
HARMA’IZ
382
350
400
450
500
550
X(nm)
FIG. 7. Light-absorption spectra of the systems given for Figs. 5 and 6. Temperature 25 f 1°C. Measurements were started from 40 to 60 min after the final pH adjustment.
serum albumins from different sources or species has been considered to constitute a very sensitive measure of differences at the respective binding sites (2, 6). Conformational differences among the albumins and/or differences in the electrochemical properties of binding groups have been assumed to account for the effects observed (2, 6). It may be noted that only for complexes involving either HSA or RSA is an inversion with pH in the sign of the main observed CD bands apparent. It is therefore unlikely that this inversion is due mainly to changes in the orientation of the transition dipole moments of bilirubin caused by ionization of chromophoric groups of the bile pigment. More likely, changes in the chirality of the bound bilirubin molecule are involved (2, 27). The present extended comparison of complexes involving serum albumins from a larger number of species confirms the high sensitivity of CD spectra as an analytical tool for differentiating among the various albumins. This sensitivity is apparently much greater in CD spectra than in corresponding light-absorption spectra (M-20) or in CD data of complexes involving various albumins and drugs or dyes (21, 22), instead of bilirubin. While there appear to be no large differences in sedimentation veloc-
AND BLAUER
ity (see above) or, most likely, also in molecular weight (10, 23) among the albumins from different vertebrate species, some differences in viscosity or titration curves have been observed between HSA and BSA (24). Also, the fluorescence properties of relaxation processesof bound bilirubin were found to vary for complexes with albumins from different species (28). Despite general similarities in amino acid composition (29, the large variations observed in the CD spectra of the various bilirubin-albumin complexes may reflect certain differences in the primary structure of the various albumins (23), which in turn could affect the tertiary structure and hence the nature of the binding sites of the various albumins. As already mentioned, possible heterogeneity in the various albumins (see, for example, ref. 26) may account, in some cases, for the differences in binding observed, since a specific fraction may bind bilirubin selectively. In the previously investigated systems involving HSA and BSA complexes at various pH values, the main CD bands near 460-470 nm and 405-425 nm, respectively, could be resolved fairly well into two Gaussian curves of opposite sign. However, the rotatory strengths of these two bands were not quite equal in many cases, indicating mixing with other states or additional contributions to the optical activity (2). Many of the visible-range CD bands given in the present and previous work (6) for various bilirubin-albumin complexes show a more complicated pattern, e.g., complexes of GSA at pH 7.4 and 4.0, of RSA at pH 4.0 (ref. 6, Figs. 1 and 2) or of PSA at pH 7.4 (Fig. 6). It seems possible that equilibria between two or more types of complexes are observed in some cases. For the GSA and RSA complexes at pH 7.4, resolution of the experimental CD and light-absorption spectra into Gaussian curves has now been carried out. In the case of the bilirubin-RSA complex, at least three Gaussians are required in the range 300-550 nm, with fair agreement in position and band width of the resolved CD and lightabsorption bands. If exciton bands of equal and opposite rotatory strength are taken into account, an additional positive Gaus-
BILIRUBIN-SERUM
sian curve is required in the CD spectrum. In the case of the GSA complex at pH 7.4, at least four Gaussian curves are necessary for reasonable resolution in the same wavelength range. However, there is less agreement between the parameters of CD and light-absorption bands than in the previous case. A more detailed treatment of the resolution and interpretation of observed spectra is under investigation. ACKNOWLEDGMENTS The skillful technical assistance and of Mr. P. Yanai is appreciated. supported by a Grant (No. 111772) Volkswagenwerk.
of Mrs. J. Silfen This work was from the Stiftung
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ALBUMIN
SPECIES
383
pp. 143-178, Academic Press, New York. 11. SCHMID, R., DIAMOND, I., HAMMAKER, L., AND GUNDERSEN, C. B. (1965) Nature (London) 206, 1041-1043. 12. JACOBSEN, J. (1969) FEBS Lett. 5, 112-114. 13. BLAUER, G. AND KING, T. E. (1970) J. Biol. Chem. 245, 372-381. 14. WENNBERG, R. P. AND COWGER, M. L. (1973) Clin. Chin. Acta 43, 55-64. 15. Hsu, M. C. AND WOODY, R. W. (1971) J. Amer. Chem. Sot. 93, 3515-3525. 16. MOSCOWITZ, A., KRUEGER, W. C., KAY, I. T., SKEWES, G., AND BRUCKENSTEIN, S. (1964) Proc. Nat. Acad. Sci. USA 52, 1190-1194. 17. BLAUER, G. AND ZVILICHOVSKY, B. (1973) Zsr. J. Chem. 11.435-443. 18. PEMBERTON, J. R. AND DE JONG, J. (1971)Anal. Biochem. 43, 575-581. 19. WITIAK, D. T. AND WHITEHOUSE, M. W. (1969) Biochem. Pharmacol. 18, 971-977. 20. BAXTER, J. H. (1964) Arch. Biochem. Biophys. 108,375-383. 21. CHIGNELL, C. F. (1969) Mol. Pharmacol. 5, 244252,455-462. 22. BRAND, J. G. AND TORIBARA, T. Y. (1973) Biochem. Biophys. Res. Commun. 52, 511516. 23. PUTNAM, F. W. (1965) The Proteins (Neurath, H., ed.), 2nd edn, Vol. 3, pp. 154-267, Academic Press, New York-London. 24. STEINHARDT, J., KRIJN, J., AND LEIDY, J. G. (1971) Biochemistry 10, 4005-4014. 25. FOSTER, J. F. (1967) Encyclopedia of Biochemistry (Williams, R. J. and Lansford, E. M., Jr., eds.), p. 20, Reinhold, New York. 26. WONG, Kc P. AND FOSTER, J. F. (1969) Biochemisty 8,4104-4108. 27. BLAUER, G. AND WAGNIPRE, G. (1975) J. Amer. Chem. Sot. 97, 1949-1954. 28. CHEN, R. F. (1974)Arch. Biochem. Biophys. 160, 106-112.