Spectral characteristics of bilirubin-bovine albumin complexes

doi:10.1016/0009-8981(73)90117-4

Clinica Chimica Acta

Volume 43, Issue 1, 10 January 1973, Pages 55-64

https://doi.org/10.1016/0009-8981(73)90117-4 Get rights and content

Abstract

Spectral characteristics of bilirubin-bovine albumin complexes have been examined. In the presence of excess albumin, the absorption spectrum of the complex did not change from pH 11.5–8.5. As the pH decreased from 8.5 to 5.5, there was a concomitant shift in the absorption maximum from 472 nm to 454 nm, and the spectra intersected at an isobestic point. This transition included the physiologically important pH range and had a pK = 6.6. Between pH 4.5 and pH 3.4 a complex with maximum absorbance at 421 nm was formed and appeared to be dependent on the N-F isomer transformation of albumin.

At pH 5.1, there was spectral evidence that 1 mole of albumin binds only 1 mole bilirubin, since difference spectra of bilirubin-albumin solutions containing excess bilirubin were similar to the spectrum of unbound bilirubin. At pH 8.5, 1 mole albumin would appear to bind 1 mole bilirubin with an absorption maximum at 472 nm, although difference spectra indicated that albumin forms additional complexes with an absorption maximum at 424 nm.

References (28)

V.A. Najjar et al.
J. Biol. Chem.
(1953)
G. Blauer et al.
J. Biol. Chem.
(1970)
P.V. Woolley et al.
Arch. Biochem. Biophys.
(1970)
G.B. Odell et al.
J. Pediat.
(1969)
R.F. Chen
J. Biol. Chem.
(1967)
J.F. Foster et al.
J. Biol. Chem.
(1965)
H.A. Petersen et al.
J. Biol. Chem.
(1965)
J.D. Ostrow et al.
J. Clin. Invest.
(1963)
T.K. With
Acta Physiol. Scand.
(1945)
N.H. Martin
J. Amer. Chem. Soc.
(1949)

G.B. Odell

J. Clin. Invest.

(1959)

D. Watson

Clin. Sci.

(1962)

J. Fog et al.

Scand. J. Clin. Lab. lnvest.

(1967)

R.P. Wennberg et al.

Pediat. lies.

(1969)

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The Status of Bilirubin Measurements in U.S. Laboratories: Why Is Accuracy Elusive?
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Citation Excerpt :
Calculations are simplified if the 2 wavelengths are isosbestic for hemoglobin; such wavelengths are 454 and 540 nm.12 Such methods should be calibrated with UBIL in human serum because all bilirubin in blood specimens from healthy neonates is practically unconjugated, and the absorptivities of UBIL in other protein matrices, particularly bovine serum, human serum albumin, or bovine serum albumin, are different from that in human serum.13,14 A special case of direct spectrophotometry, available only in the Vitros 5,1 FS (Ortho Clinical Diagnostics) clinical analyzer, simultaneously measures the sum of mono- and diglucuronide, and UBIL.15
In 2003, the Chemistry Resource Committee of the College of American Pathologists introduced a new specimen in the Neonatal Bilirubin Survey. The specimen, consisting of human serum enriched with unconjugated bilirubin and thus resembling a clinical specimen, brought about an improvement in the accuracy of the measurement of bilirubin by laboratories participating in the Neonatal Bilirubin Survey. There was also an improvement in the specificity of methods measuring direct bilirubin. However, persisting inaccuracies and variability in laboratory performance have been traced to calibrators consisting of bovine serum spiked with unconjugated bilirubin and ditaurobilirubin; bovine serum causes underestimation of both bilirubins by 8 major chemical analyzers. To eradicate inaccuracy calibrators and Survey specimens should be made in human instead of bovine serum.
Influence of succinylation of bovine serum albumin on its conformation and bilirubin binding
1987, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
In order to investigate the role of lysine residues in the interaction of bilirubin with bovine serum albumin, five succinylated preparations of albumin, namely: 23%, 39%, 49%, 55% and 87%, were prepared, and their conformational and bilirubin-binding properties were studied by the techniques of gel filtration, ultraviolet and visible spectroscopy, and fluorescence quenching. Gel filtration experiments performed at pH 7.0 and ionic strengths 0.15 and 1.0 suggested that the albumin molecule undergoes gradual disorganization with increase in succinylation. The Stokes radius and frictional ratio at ionic strength 0.15 increased from 3.7 nm and 1.36, respectively, for the native protein to 6.3 nm and 2.26 for maximally (87%) succinylated albumin. Interestingly, increase in ionic strength to 1.0 caused significant refolding in succinylated preparations as evidenced by a decrease in Stokes radius and frictional ratio (5.3 nm and 1.90 for 87% succinylated albumin). Progressive succinylation produced a steady decline in the intensity of bilirubin-induced fluorescence quenching, and in the visible spectral changes of the bilirubin-albumin complex at 480 nm. Both of these changes had a good correlation with increase in Stokes radius. Increase in ionic strength to 1.0 produced a significant reversal in these properties. From these results we conclude that probably none of the surface lysine residues is involved in bilirubin-albumin interaction, and that if lysine residues are involved in this interaction they must be buried in the protein interior.
The kinetics of the reaction between bovine serum albumin and bilirubin a second look
1982, Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
The kinetics of the reaction between bilirubin and bovine serum albumin have been re-investigated at 20°C. The resuits of previous authors concerning both human and bovine serum albumin were largely confirmed, namely the existence of two first-order configurational changes after a primary complex is formed in a fast, bimolecular step. From the kinetic behaviour it is concluded that this primary complex is present in non-negligible concentration after equilibrium is reached, and that it exchanges bilirubin with the surroundings with a rate constant of at least 23 s⁻¹. This also means that the secondary complex, and, possibly, also the final product, are in dynamic equilibrium with each other and with the primary complex, and, therefore, only the sum of their formation and dissociation rate constants can be measured. The dependence of the two observable relaxation times on pH does not parallel the N → B transition. On the other hand, a pH jump between 7.4 and 9.0 is assumed to monitor the N → B transition, both the free albumin and the complex in its various forms undergoing this transition at identical rates. This transition, although influencing the absorptivity of the complex, was found not to influence the strength of the binding site.
Competitive interaction of biliverdin and bilirubin only at the primary bilirubin binding site on human albumin
1981, Analytical Biochemistry
The binding of biliverdin-IXα by human albumin and serum was quantitated, using three different binding techniques, to study the effects of biliverdin on bilirubin-albumin binding. The apparent equilibrium association constants (K ± SD) and binding capacities (n) of defatted albumin, pooled adult sera, and pooled umbilical cord sera for biliverdin are: K = 1.3 ± 2 × 10⁶m⁻¹, n = 1.00; K = 13.0 ± 3 × 10⁶m⁻¹, n = 0.90; and K = 6.8 ± 0.1 × 10⁶m⁻¹, n = 0.85, respectively. Although bilirubin binds at more than one albumin site, competitive studies showed that biliverdin binds only at the primary (highest affinity) bilirubin site. Sulfisoxazole, previously thought to compete with bilirubin for the primary binding site, was found to displace bilirubin from both primary and secondary bilirubin binding sites. Biliverdin, because of its specific binding and spectral characteristics, could be a useful probe for determining the capacity of the primary bilirubin-albumin binding site.
Thermodynamics and kinetics of the neutral transition of human serum albumin, monitored by the spectral change of bound bilirubin
1980, BBA - Protein Structure
Spectral changes in the complex of human serum albumin with bilirubin in the neutral pH range are related to conformational alterations of the protein. The transformation responds to changes in temperature and concentration of calcium ions in a similar fashion as the N-B transition of albumin.
The kinetics of the structural changes are studied. The spectral shift from high to low, as well as from low to high pH, is presented in a model consisting of three relaxation steps.
Determination of association and dissociation rate constants for bilirubin-bovine serum albumin
1975, Archives of Biochemistry and Biophysics
The association rate constant for the binding of bilirubin to bovine serum albumin has been determined in a continuous-flow experiment. The value obtained is 0.9 x 10⁶m⁻¹S⁻¹. Furthermore the dissociation rate constant is determined from the rate of the peroxidase-catalyzed oxidation of bilirubin in a bilirubin-albumin solution. This figure is 3.1 × 10⁻²s ¹. Calculation of the apparent binding equilibrium constant from the two rate constants gives 2.9 x 10⁷m⁻¹. The above mentioned peroxidase oxidation has also been used for a direct estimation of the binding equilibrium constant giving 2.7 × 10⁷m⁻¹. All experiments are carried out at 36 °C and pH 7.4.

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^∗: M.L.C.: Department of Chemistry, State University of New York at Albany, 1400 Washington Avenue, Albany N.Y. 12203.

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Spectral characteristics of bilirubin-bovine albumin complexes

Abstract

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Pediat.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Clin. Invest.

Acta Physiol. Scand.

J. Amer. Chem. Soc.

J. Clin. Invest.

Clin. Sci.

Scand. J. Clin. Lab. lnvest.

Pediat. lies.