Noninvolvement of surface lysine residues of bovine serum albumin in bilirubin binding

Interaction of bilirubin with bovine serum albumin and its five succinylated forms was studied using fluorescence spectroscopy at two different ionic strengths i.e., 0.15 and 1.0 respectively. Affinity constant was found to be 1.8 x 10(7) litres/mole at 0.15 ionic strength which decreased to 4.4 x 10(6) litres/mole after 87% succinylation. On increasing ionic strength to 1.0, there was a slight decrease in affinity constant for native albumin. However, affinity constant remained same in 55 and 87% modified albumins at high ionic strength. These results suggest noninvolvement of surface lysine residues in bilirubin albumin complex.     

Influence of succinylation of bovine serum albumin on its conformation and bilirubin binding

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.     

Chiroptical Properties of Bilirubin-Serum Albumin Binding Sites

Although the interactions between bilirubin and serum albumin are among the most studied serum albumin-ligand interactions, the binding-site location and the participation of bilirubin-serum albumin complexes in pathological and physiological processes are under debate. In this article, we have benefited from the chiral structure of bilirubin and used CD spectroscopy to characterize the structure of bilirubin bound to human and bovine serum albumins. We determined that in a phosphate buffer at pH 7.8 there are three binding sites in both human and bovine serum albumins. While the primary binding sites in human and bovine serum albumins bind bilirubin with P- and M-helical conformations, respectively, the secondary binding sites in both albumins bind bilirubin in the P-helical conformation. We have shown that the bonding of bilirubin to the serum albumin matrix is a more favorable process than the self-association of bilirubin under the studied conditions, with a maximum of three bound bilirubins per serum albumin molecule. Although bilirubin bound to the primary binding site has attracted the most attention, the presented results have documented the impact of the secondary binding sites which are relevant in the displacement reactions between BR and drugs and in the phenomena where bilirubin plays antioxidant, antimutagenic, and anti-inflammatory roles. Chirality 00:000000, 2013. © 2013 Wiley Periodicals, Inc.     

The fatty acid analogue 11-(dansylamino)undecanoic acid is a fluorescent probe for the bilirubin-binding sites of albumin and not for the high-affinity fatty acid-binding sites.

1. The fluorescent fatty acid probe 11-(dansylamino)undecanoic acid (DAUDA) binds with high affinity to bovine and human serum albumin (BSA and HSA) at three sites. 2. The Kd of the primary binding site could not be determined; however, the two secondary sites appeared to be equivalent, with an apparent Kd of 8 x 10(-7) M for both BSA and HSA. 3. The spectral characteristics of DAUDA when bound to the primary site of the two albumins were different, with HSA producing a greater fluorescence enhancement and emission maximum at a shorter wavelength (480 nm) than for BSA (495 nm). 4. Displacement studies indicated that the DAUDA-binding sites were not equivalent to the primary long-chain fatty acid-binding sites on albumin, but corresponded to the bilirubin sites. Fatty acyl-CoAs also bind to the bilirubin sites, as do medium-chain fatty acids. 5. The solubility, stability and spectral properties of DAUDA make it an excellent probe for investigating the bilirubin-binding sites of albumin, particularly HSA.     

Changes of serum albumin affinity for aspirin induced by fatty acid

Saturated fatty acids such as myristic acid play an important role in the pathogenesis of cardiovascular disorders.

Using the quenching fluorescence method we examined the influence of myristate on the changes of transporting protein affinity towards aspirin—the most popular anticoagulant.

Our results showed that the presence of the myristic acid alters the stability of the anticoagulant–albumin complex. The ranges of [myristate]/[albumin] molar ratio at which the stability of drug–protein complex increases or decreases were determined. The differences in interaction between ligands and human or bovine serum albumins were identified. The competition in binding of ligands with these albumins was also described.     

Studies on the mechanism of binding of serum albumins to immobilized cibacron blue F3G A.

The interaction of Cibacron Blue F3G A-Sepharose 4B with several serum albumins was studied. Although all albumins used were fond to bind to this adsorbent, human serum albumin was bound to a far greater extent than were the others. From the results of competition experiments and n.m.r. studies of Cibacron Blue and/or bilirubin binding to human serum albumin it is proposed that the mechanism of the interaction between human serum albumin and cibacron Blue is consistent wit Cibacron Blue binding to bilirubin-binding sites. In contrast with these findings with human serum albumin, there is little or no interaction of Cibacron Blue and the bilirubin-binding sites of albumins from rabbit, horse, bovine or sheep sera, although some interaction occurs between Cibacron Blue and the fatty acid-binding sites of these proteins. Structural analogues of Cibacron Blue have been used to investigate the binding of albumins to these ligands.     

Binding of bromophenol blue to bovine serum albumin and its succinylated forms

Interaction of bromophenol blue with bovine serum albumin and its five succinylated forms was studied spectrophotometrically at three different ionic strengths, i.e. 0.04, 0.15 and 1.0 and at two different pH values, namely pH 7.0 and pH 5.0 respectively. Results showed a decrease in bromophenol blue binding on increasing succinylation at low ionic strengths. This decrease was more marked at pH 7.0 than pH 5.0. However, at both the pH values binding returned to a significant degree on increasing the ionic strength to 1.0. Succinylation also caused marked conformational changes at pH 7.0 and ionic strength 0.15 as evidenced by changes in hydrodynamic properties and reduction in antigen-antibody precipitin reaction. However, an increase in ionic strength to 1.0 or decrease in pH to 5.0 caused significant reversal in hydrodynamic parameters. These studies show that lysine residues of bovine serum albumin are not important in bromophenol blue binding.     

Structural and functional differences between fetal and adult serum albumin

The binding of bilirubin with adult of fetal human serum albumin has been studied by steady-state fluorescence emission spectroscopy. The 1:1 complex between bilirubin and the two albumin samples shows very similar fluorescence properties, as well as essentially identical accessibility of the protein-bound bilirubin to fluorescence quenchers added to the aqueous medium. The intramolecular distance between bilirubin and the single tryptophyl residue can be estimated to be 2.4 +/- 0.2 nm for both proteins by singlet-singlet energy transfer. These findings suggest that fetal and adult human serum albumin have a very similar three-dimensional structure; the different binding capacity for bilirubin displayed by the two proteins is likely to be the consequence of small differences in the physico-chemical properties of some amino acid residues close to the bilirubin binding site, as indicated by pH-titration experiments of the intrinsic albumin fluorescence.     

Modulation in the photosensitivity of albumin-bound bilirubin

Exposure of BR–albumin complexes to visible light at pH 8.0 led to a change in the fluorescence intensity at 525 nm, which was found to be different for different serum albumins. Whereas a complex of BR with human serum albumin (HSA) showed a marked increase in fluorescence upon photoirradiation, BR–sheep serum albumin (SSA) complex failed to produce a marked increase. On the other hand, a complex of pig serum albumin (PSA) with BR produced a remarkable decrease in fluorescence upon photoirradiation. Equilibration of these complexes with 20 mM chloroform for 1 h resulted in alteration in the photoinduced fluorescence. These photoinduced fluorescence modulations were found to be concentration dependent. Photoirradiation of BR–HSA complex led to a significant decrease in the positive CDCEs of the bisignate CD spectra in a time dependent manner that can be reconciled, to a significant extent, in the presence of chloroform. Taken together, all these results suggest that chiroptical properties/stability of albumin-bound BR varies with albumin species, protein concentration and the presence of chloroform.     

Interaction of bilirubin with brain capillaries and its toxicity

The interaction of bilirubin with isolated brain capillaries, and the effect of bilirubin on the uptake of 2-deoxyglucose by the capillaries were investigated with 1-month-old Sprague-Dawley rats. The binding of bilirubin to the brain capillaries was observed only at a molar ratio of bilirubin to bovine serum albumin higher than 1.0. An absorption spectrum with a microspectrophotometer of the bilirubin-capillary complex showed a broad absorption maximum from 425 to 440 nm with a shoulder near 490 nm, but no shoulder was observed in the case of the bilirubin emulsion. The bilirubin binding activity was dependent on pH and temperature of the medium, but was not affected by sulfhydryl blocking agents such as p-chloromercuribenzoate and N-ethylmaleimide. Bilirubin saturation kinetics gave an apparent K_m for bilirubin of 61.7 μM. Release of the bilirubin from the brain capillaries to the medium was observed at 37°C but not at 4°C. The uptake of 2-deoxyglucose by the isolated brain capillaries was inhibited by bilirubin in a noncompetitive manner, giving an apparent K_i for the pigment of 137 μM.These results suggest that bilirubin may be responsible for the decreased 2-deoxyglucose uptake in the brain capillaries by disturbing the membrane structure of the capillary endothelial cells.     

A study of the interaction of avidin with 2-anilinonaphthalene-6-sulfonic acid as a probe of the biotin binding site

The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosower's Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.     

A comparative analysis on the binding characteristics of various mammalian albumins towards a multitherapeutic agent,pinostrobin

The interaction of pinostrobin (PS), a multitherapeutic agent with serum albumins of various mammalian species namely, goat, bovine, human, porcine, rabbit, sheep and dog was investigated using fluorescence quench titration and competitive drug displacement experiments. Analysis of the intrinsic fluorescence quenching data revealed values of the association constant, K_a in the range of 1.49 – 6.12 × 10⁴ M⁻¹, with 1:1 binding stoichiometry. Based on the PS–albumin binding characteristics, these albumins were grouped into two classes. Ligand displacement studies using warfarin as the site I marker ligand correlated well with the binding data. Albumins from goat and bovine were found to be closely similar to human albumin on the basis of PS binding characteristics.     

Interaction of rabbit hemopexin with bilirubin

The interaction of hemopexin with bilirubin was characterized by spectrophotometric, fluorimetric and circular dichroic techniques. Hemopexin rapidly forms an equimolar complex with libirubin that has an apparent dissociation constant Kd, of 7.5.10(-7) M. The association alters the absorption band of bilirubin near 150 nm, quenches the fluorescence of tryptophan residues of hemopexin, enhances the fluorescence of bilirubin, and induces strong ellipticity extrema in bilirubin of --60 . 10(3) deg . cm2 . dmol-1 at 465 nm and +70 . 10(3) deg . cm2 . dmol-1 at 415 nm. However, the conformation-sensitive ellipticity aband at 231 nm of hemopexin is not altered. In displacement experiments using circular dichroism, heme readily replaced bound bilirubin, indicating that bilirubin and heme are bound at the same site on hemopexin. Even at molar ratios of hemopexin to albumin of 3 to 1, human serum albumin removes bilirubin from hemopexin. Hemopexin is thus unlikely to have a role in the transport of bilirubin in serum.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

Behavior of various mammalian albumins towards bilirubin binding and photochemical properties of different bilirubin-albumin complexes

Bilirubin (BR) binding properties of serum albumins from different mammalian species viz. human (HSA), equine (ESA), dog (DSA) and guinea pig (GPSA) were studied by absorption, fluorescence and CD spectroscopy. Whereas, a complex of BR with ESA produced maximum change, GPSA–BR complex showed weaker interaction as reflected from absorption and fluorescence spectroscopic data. Conformational analysis of these albumins by near- and far-UV CD spectra suggested similar structural characteristics (both secondary and tertiary structures) for ESA and HSA, whereas, DSA and GPSA had lower amounts of secondary and tertiary structures being minimum for GPSA. Photoirradiation results of BR–albumin complexes showed GPSA-bound BR more labile compared with other complexes, whereas, BR–ESA complex was found to be more stable against photoinduced chemical changes. Taken together, all these results suggest that chiroptical properties/stability of albumin bound BR varies with albumin species.     

Chloroform-induced conformational changes in the bound pigmentin bilirubin-albumin complexes

Chloroform-induced conformational changes of bilirubin (BR) bound to different serum albumins were studied by circular dichroism (CD) and fluorescence spectroscopy. Addition of a small amount of chloroform ( approximately 20 mM) to a solution containing 20 microM albumin and 15 microM BR changed the sign order and magnitude of the characteristic CD spectra of all BR-albumin complexes except BR-PSA complex which showed abnormal behavior. Monosignate negative CD Cotton effects (CDCEs) of BR complexed with SSA, GSA and BuSA were transformed into bisignate CDCEs in presence of chloroform akin to those exhibited by chloroform free solution of BR-HSA complex, indicating that the pigment acquired right handed plus (P) chirality when chloroform was added to these complexes. Bisignate CD spectra of BR complexed with HSA and BSA showed complete inversion upon addition of chloroform corroborating earlier findings. On the other hand, changes observed with BR-RSA complex were slightly different showing an additional CD band of weak intensity centered around 390 nm though inversion of CDCEs was similar to that of BR-HSA complex. Monosignate CD spectra of BR-PSA complex also showed three CD bands occurring at 409, 470 and 514 nm after chloroform addition. These results indicated significant but different effects of chloroform on the conformation of bound BR in BR-albumin complexes which can be ascribed to the changes in the exciton chirality of bilirubin probably due to altered hydrophobic microenvironment induced by the binding of chloroform at or near the ligand binding site. Chloroform severely quenched the intrinsic tryptophan fluorescence of the protein and shifted the emission maxima towards blue region in all the albumins except PSA. However, quantitative differences in both quenching and blue shift were noted in different serum albumins. This suggests that chloroform probably binds in the close vicinity of tryptophan residue(s) located in subdomain(s) IIA or IB and II both. The fluorescence of BR-albumin complexes was also found to be sensitive to the presence of a small amount of chloroform. But the changes observed in the fluorescence of the bound pigment in presence of chloroform were less marked as compared to the changes in the intrinsic fluorescence of protein per se. Taken together, these results suggest that there is at least one conserved site for chloroform binding in all these albumins which is at or near the BR binding site.     

Characterization of the fluorescence of the antitumor agent, mitoxantrone

Studies have been conducted on the absorption, fluorescence excitation and fluorescence emission of mitoxantrone, an important antineoplastic agent. Mitoxantrone has been found to fluoresce with excitation maxima at 610 and 660 nm and emission maximum at 685 nm. Further characterizations of the fluorescence were undertaken to study its usefulness in biological studies. Mitoxantrone fluorescence intensity is altered by pH and the emission spectrum is red-shifted by DNA. Furthermore, the fluorescence polarization is enhanced by DNA, confirming the binding of the antitumor agent to DNA. The fluorescence spectra are slightly modified by changes in ionic strength and the addition of albumin. Data establishing the usefulness of fluorescence to measure serum concentrations in the range of 0.0 to 100 nM are presented. Such determinations can distinguish serum mitoxantrone from its non-fluorescent primary metabolite.     

Stereoselective kinetics of warfarin binding to human serum albumin: effect of an allosteric interaction

Kinetic and equilibrium binding studies were performed on the interaction of warfarin enantiomers with human serum albumin (HSA) in the absence and presence of lorazepam acetate (LoAc) enantiomers. Binding kinetics were followed by recording changes in the fluorescence of warfarin upon binding to HSA using the stopped-flow technique. The binding of (R)-warfarin displayed an exponentially increasing fluorescence, satisfying the two-step mechanism reported previously for the racemate, i.e., a diffusion controlled pre-equilibrium is followed by a slower rearrangement of the complex. In the case of (S)-warfarin, the signal was biphasic: a fast fluorescence enhancement was followed by a slow decline. The different kinetic features indicate that the equilibrium conformations of the [(S)-warfarin-HSA] and [(R)-warfarin-HSA] complexes are achieved via different mechanisms. The phenomenon was seen in buffers of different pH and compositions. Equilibrium binding measurements indicated significantly lower molar intrinsic fluorescence for the (S)-warfarin complex, suggesting differences in the microenvironments of the bound enantiomers. In the presence of (S)-LoAc, the allosterically enhanced binding of (S)-warfarin manifested itself in accelerated relaxation kinetics. In accordance with the low molar intrinsic fluorescence determined for the stable ternary complex, the amplitude of the decline in fluorescence became larger.     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.     

Investigations of interaction mechanism and conformational variation of serum albumin affected by artemisinin and dihydroartemisinin

In this work, binding interactions of artemisinin (ART) and dihydroartemisinin (DHA) with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated thoroughly to illustrate the conformational variation of serum albumin. Experimental results indicated that ART and DHA bound strongly with the site I of serum albumins via hydrogen bond (H-bond) and van der Waals force and subsequently statically quenched the intrinsic fluorescence of serum albumins through concentration-dependent manner. The quenching abilities of two drugs on the intrinsic fluorescence of HSA were much higher than the quenching abilities of two drugs on the intrinsic fluorescence of BSA. Both ART and DHA, especially DHA, caused the conformational variation of serum albumins and reduced the α-helix structure content of serum albumins. DHA with hydrophilic hydroxyl group bound with HSA more strongly, suggesting the important roles of the chemical polarity and the hydrophilicity during the binding interactions of two drugs with serum albumins. These results reveal the molecular understanding of binding interactions between ART derivatives and serum albumins, providing vital information for the future application of ART derivatives in biological and clinical areas.