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CHEMISTRY
Vol. 254, No. 2, Issue of January 25, pp. 391-400, 1979
Printed in U.S.A.
Binding and proximity relationships of hydrophobic the physiological carrier of fatty acids, lysolecithin, and bili-
ligands on human serum albumin have been studied rubin, and it also carries many hydrophobic drugs. Several
using absorption, fluorescence, circular dichroism, and mammalian albumins have recently been sequenced (Behrens
electron paramagnetic resonance spectroscopy. The li- et al., 1975; Brown et al., 1971). Based on sequence data,
gands studied were bilirubin, two conjugated linear analysis of peptide fragments, and hydrodynamic measure-
polyene fatty acids, cis-parinaric acid and cis-eleo- ments, a partial domain model for the tertiary structure of the
stearic acid, and three nitroxide derivatives of stearic protein has recently been proposed (Anderson and Weber,
acid with doxyl groups at positions 5, 10, and 12, re- 1969; Pederson and Foster, 1969; Brown, 1976). According to
spectively. Binding of polyene fatty acids was moni-
391
392 Ligand Binding to Albumin
duced chromophore circular dichroism. When 2 mol of PnA respectively. The absorption maximum of cEsA exhibits a
are bound per mol of protein (V = 2), a negative component of similar shift upon binding, as shown in Fig. 2C. The peaks of
the circular dichroism is observed, which was attributed to the cEsA absorption spectrum are not sufficiently resolved to
excitonic ligand-ligand interaction (Sklar et al., 1977c). The permit measurement of valley to peak ratios. These results
emission spectrum of tryptophan overlaps the absorption are very similar to those previously reported for bovine serum
spectra of cPnA and cEsA, and this feature allowed measure- albumin (Sklar et al., 1977c).
ment of distances from the two tryptophans to the first fatty Circular Dichroism Spectra of cPnA Bound to Human
acid binding site by application of the Fijrster-Dexter theory Serum Albumin-cPnA is not optically active. An intense CD
of energy transfer. is observed in the region of cPnA absorption for several
In this study we extend these absorption, circular dichroism, cPnA.protein complexes (Sklar et al., 1975, 1977c). Fig. 3
and fluorescence measurements to human serum albumin. To shows this induced CD for the cPnA. HSA complex with V =
supplement this information, we also employ electron para- 1 and V = 2. The CD of an equal molar HSA-oleic acid solution
magnetic resonance spectroscopy of nitroxide fatty acids has been subtracted from the CD of the complex. The absorp-
bound to human serum albumin and quenching of the fluo- tion spectrum for V = 2 and the ratio AE/E are also shown for
rescence of cPnA by nitroxide fatty acids. These measure- comparison. The absorption spectrum for V = 1 is very similar
ments permit determination of binding affinities under various to that for V = 2.
conditions, and permit conclusions regarding proximity of Several features of these CD spectra are noteworthy. When
ligands to each other and to the single tryptophanyl residue V I: 1, the CD is entirely positive and has the same general
of human serum albumin. features as the absorption spectrum. The magnitude of this
Albumin carries bilirubin, the breakdown product of heme, positive CD is very similar to that observed for the cPnA. BSA
to the liver for conjugation with glucuronic acid and subse- complex (Sklar et al., 1977c). A comparison of the absorption
quent excretion. Albumin has one very strong binding con- and CD spectra for the cPnA. HSA complex shows that the
stant ( -lo8 M-) and two weaker binding constants for biliru- CD is blue-shifted relative to the absorption and that the
See Miniprint
TABLE I
Association constants for cPnA and human serum albumin
Binding of cPnA to human serum albumin was determined by the
method of fluorescence enhancement as described under Materials
and Methods. Binding constants were determined by a nonlinear
least squares fit to the coefficients of Equation 7. The buffer is
described under Materials and Methods. The RMS error is the root
mean square deviation between the measured values of V and those
calculated with Equation 7 and the binding constants given. The
enthalpy change on binding to the first site is calculated from the
23C and 37C data to be 2.5 kcal/mol and the entropy change is
roughly 28 Cal. mol- deg-.
5C 23C 37C
liters/m01
Kl 1.3 x los 1.1 x lo8 9.1 x lo7 FIG. 6. Spectral overlap between human serum albumin emission
KZ 3.0 x lo7 2.4 x lo7 2.8 x 10 and the absorption of polyene fatty acids. The broad solid curve
l-6 2.6 x lo7 1.9 x lo7 1.5 x lo7 shows the emission spectrum of human serum albumin upon excita-
K4 3.7 x lo6 6.0 x 10 5.1 x 10 tion at 294 nm. The dashed curve and the three-peaked solid curve
KS 2.8 x 10 2.1 x 10 4.6 X 10 are the absorption spectra of cPnA and cEsA, respectively. The
K6 2.6 x 10 1.3 x lo6 1.0 x lo6 shaded areas indicate that there is sizable spectral overlap between
RMS error 0.193 0.283 0.210 human serum albumin and cPnA and much smaller spectral overlap
between human serum albumin and cEsA.
occurs for a very small range of the relative orientation bovine serum albumin, while there is sizable quenching of
variables and which requires that there is no relative orienta- human serum albumin. This difference may be ascribed in
tional motion of the chromophores during the time between part to the fact that the emission spectrum of human serum
excitation and emission. This is because the distance meas- albumin is shifted to shorter wavelengths than the bovine
urements only depend on the sixth root of K. For instance, if serum albumin emission spectrum, and spectral overlap with
K is 4 instead of 2/, the distance will be underestimated by cEsA is greater. Another reason for the relatively smaller
35%. Similarly, if K is 0.1, the distance will be overestimated degree of quenching of bovine serum albumin is that roughly
by 35%. To eliminate the possibility of large overestimates of half the emission intensity for bovine serum albumin is
distances, which occur when K is close to 0, we employed thought to be due to the tryptophanyl residue, not present in
quenching of human serum albumin fluorescence by the ni- human serum albumin, which resides in domain I. It is to be
troxide fatty acids 5 doxyl stearate (5 NS), 10 doxyl stearate expected that this tryptophanyl residue is very far from the
(10 NS), and 12 doxyl stearate (12 NS). (In our notation, the first fatty acid bound, and would not be quenched effectively.
number indicates the carbon bearing the doxyl group.) Our In both human serum albumin and bovine serum albumin,
data is in general agreement with the data of Morrisett et al. the third and fourth fatty acids quench tryptophan emission
(1975) on bovine serum albumin in two respects: 1) the better than the first two fatty acids. Both of these findings are
quenching is greatest for 5 NS and 2) quenching increases consistent with the assignment of the two strongest binding
with Y more rapidly when V = 3 or 4 than when V < 3. The 12 sites to domain III in Browns model (Brown, 1976) and
NS fatty acid is most nearly analogous to cPnA due to the subsequent binding to domain II.
position of its substituent. At all mole ratios, the quenching Quenching of the Emission of Bound cPnA by Nitroxide
by 12 NS is much less than that of eleostearic acid. The Fatty Acids-The occurrence of a negative peak in the in-
quenching by nitroxide fatty acids is isotropic and very effi- duced CD spectrum of cPnA bound to bovine serum albumin
cient for distances less than about 8 A but is inefficient for and human serum albumin has been interpreted in terms of
large distances. The low quenching of tryptophan emission ligand-ligand exciton interaction between bound fatty acids
that the third and fourth fatty acid ligands bind closer to the PnA + OLEATE
tryptophan than the first and second ligands. If the binding is
nonsequential, then the first two sites must be even further
from the tryptophan and the third and fourth sites must be
even closer. The conclusion that the first two fatty acids bind
about equally distant from the tryptophan is supported by
independent experiments which show that these two fatty
acids are close together in the complex.
The energy transfer data presented here may be compared
with the data of Sklar et al. (1977c) in their studies of bovine
serum albumin. Binding of a single oleic acid to bovine serum
albumin reduces the protein fluorescence. This effect is not
seen in human serum albumin until V is greater than 3. When
V = 1 for cEsA, there is virtually no additional quenching of
phanyl
plotted
residue
between
were determined
Apparent
energy transfer
distances polyene fatty acids and the trypto-
using the quenching
1 to 4. The orientation
Values of the relevant
efficiencies
factor I? was
spectroscopic
2
FIG. 8. Quenching
2
by nitroxide
M
MOLE
ESNS
HSA
4
been added. This kind of experiment can be used to estimate TABLE III
the relative binding affinities of these ligands for human serum Binding of combinations of nitroxide fatty acids to human serum
albumin. Absorption and fluorescence data in cases in which albumin
V ranges from 6 to 9 for oleic acid or nitroxide fatty acids Nitroxide fatty acids were added to a human serum albumin
suggest the following order of binding affinities: oleic acid zz solution and percentages of bound and unbound probe were deter-
mined by double integration and subtraction of EPR spectra using a
cPnA > 5 NS 2 10 NS >> 12 NS. PDP8E computer, and values were checked by hand calculation of
The polyene chromophore of cPnA is located toward the peak heights and comparison with standards. Calculations are derived
methyl end of the chain, and the best quenching occurs when from the suectra shown in Fig. 9.
the doxyl moiety is nearer to the COOH terminus. This Nitroxide fatty acid Unbound nitroxide fatty acid
suggests that for the fist two fatty acids bound, the arrange- %
ment is roughly antiparallel side by side as shown in Fig. 15. 5NS CO.2
Electron Paramagnetic Resonance Spectroscopy of Nitrox- 10 NS 0.8
ide-labeled Fatty Acids Bound to Human Serum 12 NS 4.0
Albumin-The EPR spectra of nitroxide-labeled fatty acids lONS+5NS 2.9
change dramatically upon transfer from buffer to binding sites lONS+ 12NS 7.2
IONS+ 10NS 12.9
on human serum albumin. Fig. 9A shows spectra of three 5NS+12NS 6.0
nitroxide fatty acids bound to human serum albumin (V 5 1). 12NS+12NS 9.4
The general features of these spectra are similar to those 5NS+5NS 0.3
observed by Morrisett et al. (1975) in their study of bovine a Each fatty acid listed is present in an equimolar ratio to human
serum albumin. The spectral lines are broadened, reflecting serum albumin at 5 X 10-S M.
the relative immobilization of the nitroxide moieties upon
binding to the protein. Superimposed on these broad lines are
much smaller sharp components due to the presence of un-
TABLE V
Binding constants for bilirubin and human serum albumin
Binding was quantified by the use of fluorescence quenching titra-
tions at different protein concentrations as shown in Fig. 14. It is
assumed that at concentrations greater than 6 X 1Om6 M the fust 2
mol are essentially all bound. As before, the nonlinear least squares
routine was used, and three binding constants were required to
produce adequate fits. The third binding constants were 8 x 105, 7 x
105, and 6 X lo5 at 5C, 23C, and 37C, respectively. These constants
are not reliable due to the absence of sufficient data points for Y > 2.
I. C.
The RMS error is described in the legend to Table I. The enthalpy
change calculated for the first binding constant from the 23C and
MOLES BILIRUBIN 37C data is roughly 8.2 kcaI/mol and the entropy change is roughly
MOLE HSA 9 cal/mol . degree.
FIG. 12. Energy transfer from human serum albumin (HSA) to 5C 23C 37C
bilirubin. Relative emission intensities at 340 nm are recorded from liters/m01
the experiment (described in Fig. 11) in which bihrubin is added to
human serum albumin (6 X 1Om6 M, filled circles). Fluorescence K1 1.3 x lo* 1.2 x lo* 6.4 x 10
intensities were corrected for inner and outer filtering, scaled relative K2 2.4 x lo6 4.9 x lo6 2.6 x lo6
to the initial value, and plotted versus the bihrubin to human serum RMS error 0.119 0.154 0.079
albumin molar ratio. The open circles and squares show the quench-
ing efficiency of bihrubin at human serum albumin concentrations of spectrum of cPnA as shown in Fig. 10. In this case the spectral
3.5 X 10e7 M and 8 X 10-s M, respectively. The data shown are for
quenching at 23C; similar curves were obtained for 5C and 37C. overlap is extremely large, and the spectral overlap integral is
1.031 X 10-13. The value of Ro213 is calculated to be 41 A. Fig.
TABLE IV 14 shows the quenching of the fluorescence of cPnA bound to
Distances from bound bilirubin to polyene fatty acids and the human serum albumin (Y = 1) as aliquots of bilirubin are
tryptophanyl residue of human serum albumin added. A similar quenching profile is observed when 2 mol of
Apparent distances between chromophores are calculated using cPnA are bound. As noted under Materials and Methods,
the transfer efficiencies derived from Figs. 12 to 14, similar curves for the quantum yields of the first 2 bound cPnA mol are identical,
the case in which V = 2 for PnA (data not shown), and Equations 1 to and this allows calculation of distances between both bound
4. Values of the pertinent spectroscopic parameters are given in the cPnA chromophores and the first two bound bilirubin chro-
text, and K* is set equal to % in each case. mophores. As shown in Table IV, in each case, the fatty acid-
Donor-acceptor p/3
bilirubin distance is large, ranging between 35 and 40 A. This
A finding is consistent with the observation (Woolley and
Tryptophan-bihrubini 27 Hunter, 1970; Berde et aL4) that the first two fatty acids
Tryptophan-bihrubinz 24 bound to human serum albumin do not cause measurable
PnAi-bihrubinl 40
PnAl-bibrubinn 37 displacement of bilirubin.
PnAz-bihrubim 40 4 C. B. Berde, F. Rasmussen, W. Benitz, J. A. Kerner, J. D. Johnson,
PnAz-bihrubinz 35
and R. P. Wennburg, in preparation.
398 Ligand Binding to Albumin
CONCLUSIONS Arvidsson, E. O., Green, F. A., and Laurell, S. (1971) J. Biol. Chem.
Binding and proximity relationships for fatty acids and 246,5373-5379
Ashbrook, J. D., Spector, A. A., Santos, E. C., and Fletcher, J. E.
bilirubin bound to human serum albumin were studied using
(1975) J. Biol. Chem. 250,2333-2338
absorption, circular dichroism, fluorescence enhancement, and Behrens, P. Q., Spiekerman, A. M., and Brown, J. R. (1975) Fed.
fluorescence energy transfer techniques with conjugated linear Proc. 34,591
polyene fatty acids. Electron paramagnetic resonance spec- Brown, J. R. (1976) Fed. Proc. 35,2141-2144
troscopy of nitroxide fatty acids and fluorescence quenching Brown, J. R., Low, T., Behrens, P., Sepulveda, P., Parker, M., and
by nitroxide fatty acids were also used to determine proximity Blakeney, E. (1971) Fed. Proc. 30, 1241
Burstein, E. A., Vedenkins, N. S., and Ivkova, M. N. (1973) Photo-
relationships. Binding and proximity relationships for biliru-
them. Photobiol. 18, 263-279
bin were monitored by fluorescence quenching. The data Chen, R. F. (1967) J. Biol. Chem. 242, 173-181
presented here generate the following picture of ligand binding Chen, R. F. (1973) in Fluorescence Techniques in Cell Biology
td albumin. There are roughtly six strong binding sites for (Thaer, A. A., and Sernetz, M., ed) pp. 273-282, Springer-Verlag,
fatty acids, with the fist two fatty acids binding more strongly New York
than the next four. The binding is very weakly temperature- Fisher, P. A., and Kern, D. (1977) J. Biol. Chem. 252,6528-6535
dependent, indicating that the major driving force for binding Fletcher, J. E., Spector, A. A., and Ashbrook, J. D. (1970) Biochem-
istry 9,4580-4587
is a positive entropy contribution. The first two fatty acids Forster, T. (1948) Ann. Phys. 437, 55-75
bind close together and further from the tryptophanyl residue Goodman, D. S. (1958) J. Am. Chem. Sot. 80,3892-3898
than the third and fourth fatty acids. Taken together with the Hubbell, W. L., and McConnell, H. M. (1971) J. Am. Chem. Sot. 93.
bovine serum albumin results of Sklar et al. (1977c), this 314-326
supports the view that the first two binding sites are located Hudson, B. S., and KohIer, B. E. (1974) Annu. Rev. Phys. Chem. 25,
in domain III, and the third and fourth fatty acids bind in 437-460
Jacobsen, J. (1969) FEBS Lett. 5, 112-114
domain II, which contain the tryptophan. Fluorescence Klotz, I. M., and Urquhart, J. M. (1949) J. Am. Chem. Sot. 71,
quenching, EPR spectroscopy, and CD spectroscopy provide 847-851
& 4.0
z
z
Y 2.0
cl
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k!
2
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