Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site

Human serum albumin (HSA) is known to exist as N (pH approximately 7), B (pH approximately 9), and F (pH approximately 3.5) isomeric forms and an equilibrium intermediate state (I) accumulate in the urea induced unfolding pathway of HSA around 4.8-5.2 M urea concentrations. These states displayed characteristic structure and functions. To elucidate the ciprofloxacin (CFX) binding behavior of HSA, the binding of ciprofloxacin with these conformational states of human serum albumin (HSA) has been investigated by fluorescence spectroscopy. The binding constant (K) for N, B, F, and I conformation of HSA were 6.92 x 10(5), 3.87 x 10(5), 4.06 x 10(5), and 2.7 x 10(5) M(-1) and the number of binding sites (n) were 1.26,1.21, 1.16, and 1.19, respectively. The standard free energy changes (DeltaGbinding(0)) of interaction were found to be -33.3 (N isomer), -31.8 (B isomer), -32 (F isomer), and -30.0 kJ mol(-1) respectively. By using unfolding pathway of HSA, domain II of HSA has been assigned to possess binding site of ciprofloxacin. Plausible correlation between stability of CFX-N and CFX-B complexes and drug distribution have been discussed. At plasma concentration of HSA fraction of free CFX, which contributes potential to its rate of transport across cell membrane, was found to be approximately 80% more for B isomers compared to N isomers of HSA. The conformational changes in two physiologically important isomers of HSA (N and B isomers) upon ciprofloxacin binding were evaluated by measuring far, near-UV CD, and fluorescence properties of the CFX-HSA complex.     

Selectivity and stereospecificity of the reactions of dichlorodiammineplatinum(II) with three purified plasma proteins

The reactions of cis- and trans-dichlorodiammineplatinum(II) (cis- and trans-DDP) with albumin and two plasma proteinase inhibitors were compared. Reaction with alpha 2-macroglobulin (alpha 2M) resulted in subunit crosslinking and loss of proteinase binding activity. The reaction also modified a receptor recognition site present on each alpha 2M subunit. While more trans-DDP was incorporated into alpha 2M than cis-DDP, cis-DDP was more effective at blocking receptor recognition, alpha 1-proteinase inhibitor was also inactivated by reaction with either cis- or trans-DDP. These reactions resulted in binding of platinum to methionine-358 at the reactive center of this inhibitor. Trans-DDP, however, was less selective and also bound to the single cysteine residue (Cys-232) of alpha 1PI. Reaction of albumin with cis-DDP resulted in incorporation of about 1 mol platinum per mol protein, and this platinum modified the single cysteine (Cys-34) in the molecule. Albumin incorporated twice as much trans-DDP, but the binding did not involve cysteine-34. In general, reactions of cis-DDP with proteins appear to be more selective than those observed for modification with the trans isomer.     

Estimation of the extent of platination and the sites of Pt binding after interaction of cis- and trans-diamminedichloroplatinum(II) with DNA and chromatin

Differences in the mode of binding of cis- and trans-diamminedichloroplatinum(II) complexes (cis and trans-DDP) with DNA and chromatin were studied with the use of [14C]methylbromphenvinphos as an alkylating agent which attacks the sites in purines bases involved also in the reaction with cis-DDP (Oliński et al., J. Biochem. Biophys. Meth., 7, 171-173, 1983). Methylation of pre-formed DDP-DNA and DDP-chromatin complexes, followed by qualitative and quantitative analysis of the methylation products in DNA hydrolysates, permitted evaluation of the distribution and extent of platination of the bases. No major differences were found between the action of the two DDP isomers on DNA. However, a significant decrease in binding of trans-DDP to adenine moieties was observed when the interaction of cis- and trans-DDP on chromatin was compared.     

Sequence analysis of twin ATP binding cassette proteins involved in translational control, antibiotic resistance, and ribonuclease L inhibition

The urea-induced unfolding of 'N' isomer (occurring at pH 7.0) and 'B' isomer (occurring at pH 9.0) of human serum albumin was studied by fluorescence and circular dichroism spectroscopic measurements. Urea-induced destabilization in different domains of both the isomers was monitored by using domain specific ligands, hemin (domain-I), chloroform, bilirubin (domain-II), and diazepam (domain-III). Urea-induced denaturation of N and B isomers of HSA showed a two-step, three-state transition with accumulation of intermediates around 4.8-5.2M and 3.0-3.4M urea concentrations, respectively. During first transition (0-4.8M urea for N isomer and 0-3.0M urea for B isomer) a continuous decrease in diazepam binding suggested major conformational changes in domain-III prior to intermediate formation. On the other hand, binding of hemin, a ligand for domain-IB and chloroform, whose binding site is located in domain-IIA remains unchanged up to 5.0M urea for N isomer and 3.0M urea for B isomer. Similarly, fluorescence intensity of Trp-214 that resides in domain-IIA remained unchanged up to the above-said urea concentrations and decreased thereafter. Absence of any decrease in hemin binding, chloroform binding, and Trp-214 fluorescence suggested the non-involvement of domain-IB and domain-IIA in intermediate formation. A significant increase in bilirubin binding prior to intermediate formation showed favorable conformational rearrangement in bilirubin binding cavity formed by loop 4 of domain-IB and loop 3 of domain-IIA. Further, a nearly complete abolishment of bilirubin binding to both isomers around 7.0M and 6.0M urea concentrations, respectively, indicated complete separation of domain-I from domain-II from each other. From these observations it can be concluded that N to B transition of human serum albumin shifted the intermediate formation towards lower urea concentration (3.0-3.4M urea for B isomer as against 4.8-5.2M urea for N isomer). Further both the intermediates were found to possess similar alpha-helical (approximately 39%) content and ligand binding properties.     

Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXalpha

Bilirubin, an insoluble yellow-orange pigment derived from heme catabolism, accumulates to toxic levels in individuals with impaired or immature liver function. The resulting jaundice may be managed with phototherapy to isomerize the biosynthetic 4Z,15Z-bilirubin-IXα to more soluble and excretable isomers, such as 4Z,15E-bilirubin. Bilirubin and its configurational isomers are transported to the liver by human serum albumin (HSA) but their precise binding location(s) on the protein have yet to be determined. To investigate the molecular details of their interaction, we co-crystallised bilirubin with HSA. Strikingly, the crystal structure—determined to 2.42 Å resolution—revealed the 4Z,15E-bilirubin-IXα isomer bound to an L-shaped pocket in sub-domain IB. We also determined the co-crystal structure of HSA complexed with fusidic acid, an antibiotic that competitively displaces bilirubin from the protein, and showed that it binds to the same pocket. These results provide the first crystal structure of a natural bilirubin pigment bound to serum albumin, challenge some of the present conceptions about HSA-bilirubin interactions, and provide a sound structural framework for finally resolving the long-standing question of where 4Z,15Z-bilirubin-IXα binds to the protein.     

顺,反铂配合物诱导生物合成金属硫蛋白的研究

给家兔皮下注射不同结构的铂配合物溶液、对肝、肾组织的可溶性组分进行凝胶和离子交换层析分离,研究了它们诱导金属硫蛋白合成的性质,发现顺铂只选择性地诱导肝脏合成ＭＴ,反铂对肝,肾都不诱导,Ｋ２ＰｔＣｌ４同时可在肝和肾脏中诱导ＭＴ的合成。并通过用Ｚｎ（ＮＯ３）２预先诱导肝和肾中合成ＭＴ,然后每日以２、４、４和８ｍｇＰｔ／ｋｇ的剂量连续皮下注射４天顺铂或反铂溶液后,在肝和肾中分离出大量的铂化硫蛋白,且用反     

Interaction of anesthetic supplement thiopental with human serum albumin

Thiopental (TPL) is a commonly used barbiturate anesthetic. Its binding with human serum albumin (HSA) was studied to explore the anesthetic-induced protein dysfunction. The basic binding interaction was studied by UV-absorption and fluorescence spectroscopy. An increase in the binding affinity (K) and in the number of binding sites (n) with the increasing albumin concentration was observed. The interaction was conformation-dependent and the highest for the F isomer of HSA, which implicates its slow elimination. The mode of binding was characterized using various thermodynamic parameters. Domain II of HSA was found to possess a high affinity binding site for TPL. The effect of micro-metal ions on the binding affinity was also investigated. The molecular distance, r, between donor (HSA) and acceptor (TPL) was estimated by fluorescence resonance energy transfer (FRET). Correlation between the stability of the TPL-N and TPL-F complexes and drug distribution is discussed. The structural changes in the protein investigated by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy reflect perturbation of the albumin molecule and provide an explanation for the heterogeneity of action of this anesthetic.     

cis-dichlorodiammineplatinum (II) as a selective modifier of the oxidation-sensitive reactive-center methionine in alpha 1-antitrypsin

Methionine 358 in the plasma protein alpha 1-antitrypsin (alpha 1AT) is an oxidation-sensitive reactive-center residue critical for proteinase-inhibitory activity. Reaction of alpha 1AT with 20 microM to 1.67 mM cis-dichlorodiammineplatinum (II) (cis-DDP) or trans-DDP afforded concentration-dependent loss of trypsin-inhibitory activity. This effect, studied by gel electrophoresis and activity assays, is essentially independent of pH over the range 4.9-8.6. Binding assays showed covalent incorporation of 1 mol of cis-DDP into each mol of alpha 1AT. cis-DDP protected a single methionine residue from oxidation and made alpha 1AT resistant to degradation by papain, which cleaves alpha 1AT at Met358. These findings strongly suggest that cis-DDP inactivates alpha 1AT by binding exclusively to its reactive-center methionine. alpha 1AT bound twice as much platinum when reacted with trans-DDP. Because carboxamidomethylated alpha 1AT incorporated nearly 1 mol of both cis- and trans-DDP, the trans isomer apparently binds to both the reactive-center methionine and to the single cysteine residue of alpha 1AT. Because of its greater selectivity, cis-DDP is the superior reagent for modification of the alpha 1AT reactive-center methionine.     

Unwinding of supercoiled DNA by cis- and trans-diamminedichloroplatinum(II): influence of the torsional strain on DNA unwinding.

The effective unwinding angle, phi, for cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP was determined by utilizing high resolution gel electrophoresis and supercoiled phi X174 RF DNA as a substrate. The effective unwinding angle was calculated by equating the reduction in mobility of the DDP-modified DNA to the removal of a number of superhelical turns. The value of the effective unwinding angle for both DDP isomers was greatest at the low levels of DDP bound and decreased with increasing amounts of unwinding agent. The cis-isomer is a better unwinding agent than is the trans-isomer, being nearly twice as effective in unwinding the supercoiled DNA at the DDP levels investigated. A comparison of the magnitude of phi below rb values of 0.005 and those at high levels of binding reveals that the extent of torsional strain in the supercoiled DNA influences the magnitude of the unwinding of the DNA by these complexes. When this method is used in the analysis of the unwinding angle for a covalently bound species on supercoiled DNA, it may provide a more reliable estimate of the magnitude of phi at high degrees of supercoiling and at low levels of modification.     

Paclitaxel-HSA interaction. Binding sites on HSA molecule

Paclitaxel (trade name Taxol) is one of the world's most effective anticancer drugs. It is used to treat several cancers including tumours of the breast, ovary and lung. In the present work the interaction of paclitaxel with human serum albumin (HSA) in aqueous solution at physiological pH has been investigated through CD, fluorescence spectroscopy and by the antibody precipitate test. Binding of paclitaxel to albumin impact on protein structure and it influences considerably albumin binding of other molecules like warfarin, heme or bilirubin. The paclitaxel-HSA interaction causes the conformational changes with the loss of helical stability of protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the paclitaxel-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilization of the warfarin binding site, which includes Trp 214, and high affinity bilirubin binding site located in subdomain IIA.     

In vivo effects of cis- and trans-diamminedichloroplatinum(II) on SV40 chromosomes: differential repair, DNA-protein cross-linking, and inhibition of replication

The mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, was investigated by using the approximately 5200 base pair (bp) chromosome of simian virus 40 (SV40) as an in vivo chromatin model. Comparative studies were also carried out with the clinically ineffective isomer trans-DDP. Although 14 times more trans- than cis-DDP in the culture medium is required to inhibit SV40 DNA replication in SV40-infected green monkey CV-1 cells, the two isomers are equally effective at inhibiting replication when equimolar amounts are bound to SV40 DNA in vivo. Since both isomers are transported into CV-1 cells at similar rates, differential uptake cannot account for the greater ability of cis-DDP to inhibit SV40 DNA replication. Rather, this result is explained by the finding that cis-DDP-DNA adducts accumulate continuously over the incubation period, whereas trans-DDP binding to DNA reaches a maximum at 6 h and thereafter decreases dramatically. We suggest that the different accumulation behavior of cis-DDP and trans-DDP on DNA is due to their differential repair in CV-1 cells. A variety of non-histone proteins, including SV40 capsid proteins but virtually no histones, are cross-linked to SV40 DNA in vivo by either cis- or trans-DDP. More DNA-protein cross-links are formed by trans-DDP than by cis-DDP at equivalent amounts of DNA-bound platinum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective protein binding of ketoprofen: Effect of albumin concentration and of the biological system

Equilibrium dialysis was used to study in vitro the enantioselective binding of R, S, and racemic ketoprofen at physiological pH and temperature in human serum albumin (HSA) (1, 20, and 40 g/liter) and in plasma. The binding of enantiomers in a racemic mixture was studied to see the effect of each isomer on the other's interaction with the protein. The free fractions were determined by high-performance liquid chromatography. The binding of ketoprofen enantiomers to albumin was enantioselective, depending on both drug and protein concentrations. Enantioselectivity was observed in plasma too but was the opposite of that in HSA at 40 g/liter. The percentage of each isomer unbound was higher in the racemic mixture than with the isomer alone. The displacement of probes specific for HSA sites I and II, studied by spectrofluorimetry, suggests that all three preparations of ketoprofen are bound mainly to site I and secondarily to site II. © 1993 Wiley-Liss, Inc.     

Interactions of a DNA molecule with coordination compounds of platinum and cobalt in solutions

The interaction of DNA with coordination compounds of divalent platinum and trivalent cobalt was studied. Complexes containing simultaneously different coordination compounds were prepared by various procedures. It was shown that the binding of trans-diaminodichloroplatinum (DDP) to DNA prevents its further complexing with cis-DDP, while in the presence of cis-DDP the possibility for trans-DDP to bind to DNA in solution remains. The study of similar DNA complexes with trans-DDP and cobalt hexamine indicated the competition for the binding sites of these compounds to the DNA molecule.     

Bilirubin binding properties of pigeon serum albumin and its comparison with human serum albumin

Binding of bilirubin (BR) to pigeon serum albumin (PgSA) was studied by absorption, fluorescence and CD spectroscopy and results were compared with those obtained with human serum albumin (HSA). PgSA was found to be structurally similar to HSA as judged by near- and far-UV CD spectra. However, PgSA lacks tryptophan. Binding of BR to PgSA showed relatively weaker interaction compared to HSA in terms of binding affinity, induced red shift in the absorption spectrum of BR and CD spectral characteristics of BR-albumin complexes. Photoirradiation results of BR-albumin complexes also showed PgSA-bound BR more labile compared to HSA-bound BR.     

Determining binding sites of drugs on human serum albumin using FIA-QCM

A simple and effective method was developed for determining binding sites of drugs on human serum albumin (HSA) by independent binding or competitive displacement of bilirubin using flow injection analysis-quartz crystal microbalance (FIA-QCM) system. Both independent and competitive bindings were entirely monitored in real time. Bilirubin as a site I-binding ligand was pre-bound to HSA sensor so as to occupy the drug-binding site I. When the model site II-binding drugs (ibuprofen, ketoprofen and flurbiprofen) were injected into the bilirubin pre-bound HSA system, the frequency continuously decreased by 6Hz, 4Hz and 5Hz, respectively, which was the same as that of their individual binding to HSA sensor. It indicated that the drug binding to site II was independent and did not interfere with bilirubin binding. However, when the model site I-binding drugs (iodipamide and magnesium salicylate) were introduced into the system, the frequency remained unchanged in the initial several minutes and then rapidly decreased by 4Hz for iodipamide and increased by 4Hz for magnesium salicylate. This phenomenon revealed site I-binding drugs competitively bound to HSA against bilirubin and displaced the pre-bound bilirubin. The results demonstrate FIA-QCM can be a valid approach for monitoring the dynamic interaction between drugs and HSA in real time further identifying drug-binding sites without the need of labels.     

Evidence for pH-dependent multiple conformers in iron(II) heme–human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Urea induced unfolding of F isomer of human serum albumin: a case study using multiple probes

The human serum albumin is known to undergo N <==> F (neutral to fast moving) isomerization between pH 7 and 3.5. The N < ==> F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher KSV of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).     

Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp–HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment–HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).