Rom. J. Biophys. 2002 12(3-4):91-96
BILIRUBIN
FLUORESCENCE. AN EXPERIMENTAL STUDY
GABRIELA
*Department of Chemistry, Faculty of Biotechnologies, University of Agricultural Sciences and Veterinary Medicine, 59, Marasesti Blvd, Bucharest 32, Romania
**Department of Biophysics, Faculty of Physics, University of Bucharest, P.O. Box MG-11, MagureleΧBucharest, Romania
Abstract.
The fluorescence of bilirubin (BR) was studied in the presence of different
concentrations of bovine serum albumin (BSA). It was also investigated the
influence of sucrose concentration on the bilirubin fluorescence.
Key words:
bilirubin, fluorescence, bovine serum albumin, neonatal jaundice.
INTRODUCTION
The excited states of bilirubin (Fig. 1) are of
great importance because of their relevance for the understanding of the
molecular mechanisms implied in the phototherapy of newborn with physiologic
jaundice [5]. After the excitation of BR from the ground state (S0)
to its first excited singlet (S1) it can decay to S0 with
fluorescence emission.
Fig. 1 - Electronic
excitation of ground state bilirubin (S0) to its first excited
singlet (S1) that can decay to S0 with fluorescence.
It is to be noticed that the attempt of Alexander Cu
and collab. [3] to evidence the fluorescence of free
bilirubin in solution did not succeed. Later, it was phototherapy that
emphasized that BR gives a fluorescence emission only when bound to different
serum albumins (human, bovine, dog, goat, pig) [1, 2]; it was observed an
enhancement of the fluorescence emission as the viscosity of the solution
increases [4]. The detection of the fluorescence of BR bound to serum albumins
is relevant for the efficiency of the newborn jaundice phototherapy. The
irradiation with visible light results in bilirubin degradation. The
photoproducts do not emit at the same l as BR (l = 550 nm).
MATERIALS AND
METHODS
All
reagents were of analytic purity. Bilirubin and bovine serum albumin were
purchased from
The
fluorescence spectra were recorded with an Aminco-Bowman spectrofluorimeter, at
room temperature, with excitation at l = 465 nm. Data acquisition in digital format
was performed with AUTOLAB software running on a 486-type PC linked to the
spectrofluorimeter.
RESULTS
A typical fluorescence spectrum of BR (2 mM) with 15 mM BSA in solution is presented in Figure 2.
Fig. 2. Fluorescence spectrum of bilirubin
(2 mM) in
the presence of BSA (15 mM).
BR
FLUORESCENCE WITH 1.5 mM BSA
There were prepared solutions of BR in dioxane and
mixed in phosphate-buffered solutions, pH = 7.5, with 1.5 mM BSA. The final
concentrations in the test-tubes were: 0.5, 1, 2, 4, 6, 10, and 14 mM. The fluorescence emission
(l = 550
nm), in arbitrary units (A.U.), function of the BR concentration is represented
in Figure 3.
Fig. 3. Fluorescence
emission of BR in solution with 1.5 mM
BSA.
BR
FLUORESCENCE WITH 15 mM BSA
The solutions were prepared in the same conditions,
but with a BSA concentration of 15 mM. The results are shown in Figure 4.
Fig. 4. Fluorescence emission of
BR in solution with 15 mM BSA.
The comparative results for both BSA concentrations
are presented in Fig. 5.
Fig. 5. Fluorescence
emission of BR for both BSA concentrations; ■ = 1.5 mM BSA: ¨= 15 mM BSA
BR
FlUORESCENCE WITH 15 mM BSA IN THE PRESENCE OF VARIABLE SUCROSE CONCENTRATION
To the solutions prepared as above, with a constant
BR concentration (2.5 mM with 15 mM BSA, there were added various amounts of sucrose (20, 40, 60, 80%).
The fluorescence spectra recorded from these solutions are shown in Fig. 6.
Fig. 6. Fluorescence spectra of
BR (2 mM) in 15 mM BSA solutions, at different sucrose concentrations: 20, 40, 60 and
80%, presented in the order of increasing amplitudes.
The fluorescence emission is enhanced as the sucrose
concentration increases, as it can be seen in Figure 7.
Fig. 7. Fluorescence emission of
BR (l = 550
nm) function of sucrose concentration.
DISCUSSION
As
it results from Figures 3 5, at both BSA concentrations, above a certain BR
concentration, the fluorescence emission remains constant. Taking into account previous
studies [3, 6] and the fact that, at room temperature, the bilirubin has
fluorescence emission only bound to serum albumins [1] it results that the
number of binding sites of BR to serum albumin is limited. By this way, we can
explain why, in vivo, when the BR concentration in the blood exceeds a certain
value, the surplus of BR cannot bind the serum albumin any more. The free BR,
being liposoluble, can enter through the lipid hilayer of different cells. It
results in severe damages, especially in nervous cells, as in the case of
newborn jaundice. The BR liposolubility explains also its interaction with the
erythrocyte membrane [7, 8].
In
the same figures, it can also be seen that the fluorescence emission enhances
with the increase of the amount of BR bound to the serum albumin.
Because
at equal BR concentrations, the fraction of bound BR is greater in the solution
containing more BSA, we could expect that the fluorescence emission would be
greater in this case. But, we can see in Figure 5, this is the case only until
2 mM
In
Figures 6 and 7, we can see that, in spite of the fact that the shape of the
curves is the same, the fluorescence emission is
enhanced as the sucrose concentration increases. Because all the other
conditions were the same as before, the only difference consists in an
increased viscosity at greater sucrose concentrations.
In
conclusion, from our study evidences results that the fluorescence emission of
BR supposes its binding to serum albumin and it is enhanced as the medium
viscosity increases.
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