The drug binding site of human alpha1-acid glycoprotein: insight from induced circular dichroism and electronic absorption spectra

Human alpha(1)-acid glycoprotein (AGP) is an important drug binding plasma protein which affects pharmacokinetical properties of various therapeutic agents. For the first time, interpretation of the induced circular dichroism (ICD) spectra of drug-AGP complexes is presented yielding valuable information on the protein binding environment. ICD spectra were obtained by novel ligands of which AGP induced optical activity have never been reported (primaquine, mefloquine, propranolol, terazosin, carbamazepine, rhodamine B) and by re-investigation of ICD spectra of protein-bound drugs published earlier (chlorpromazine, dipyridamole, prazosin). Spectroscopic features of the ICD and absorption bands of drugs combined with native AGP indicated chiral non-degenerate exciton coupling between the guest chromophore and the indole ring of an adjacent tryptophan (Trp) residue. Results of additional CD experiments performed by using recombinant AGP mutants showed no changes in the ligand binding ability of W122A in sharp contrast with the W25A which was unable to induce extrinsic CD signal with either ligand. Thus, these findings unequivocally prove that, likely via pi-pi stacking mechanism, Trp25 is essentially involved in the AGP binding of drugs studied here as well as of related compounds. Survey of the AGP binding data published in the literature support this conclusion. Our results provide a fast and efficient spectroscopic tool to determine the inclusion of ligand molecules into the beta-barrel cavity of AGP where the conserved Trp25 is located and might be useful in ligand-binding studies of other lipocalin proteins.     

Circular dichroism and absorption spectroscopic data reveal binding of the natural cis-carotenoid bixin to human alpha1-acid glycoprotein

Using circular dichroism (CD) and electronic absorption spectroscopy techniques, interaction of the natural dietary cis-carotenoid bixin with an important human plasma protein in vitro was demonstrated for the first time. The induced CD spectra of bixin obtained under physiological conditions (pH 7.4, 37 degrees C) revealed its binding to the serum acute-phase reactant alpha(1)-acid glycoprotein (AGP), a member of the lipocalin protein family. Spectral features of the extrinsic Cotton effects of bixin suggested the inclusion of a single, chirally distorted ligand molecule into the asymmetric protein environment. Compared with the absorption spectra obtained in ethanol and benzene, the strong red shift of the main absorption peak of AGP-bound bixin indicated that the proposed binding site was rich in aromatic residues, and also suggested that hydrophobic interactions were involved in the binding. Using the data obtained from the CD titration experiments, the association constant (Ka=4.5x10(5)M-1) and stoichiometry of the binding (0.15) were calculated. The low value of the stoichiometry was attributed to the structural polymorphism of AGP. To the authors' knowledge, the current study represents the first human lipocalin protein for which carotenoid binding affinity has been explored in vitro with these techniques.     

Progesterone binding to the tryptophan residues of human alpha1-acid glycoprotein

Binding studies between progesterone and alpha1-acid glycoprotein allowed us to demonstrate that the binding site of progesterone contains one hydrophobic tryptophan residue and that the structure of the protein is not altered upon binding. The data obtained at saturated concentrations of progesterone clearly reveal the type of interaction at physiological levels.     

Selective binding of imatinib to the genetic variants of human alpha1-acid glycoprotein

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia. Its strong plasma protein binding referred to alpha1-acid glycoprotein (AGP) component was found to inhibit the pharmacological activity. AGP shows genetic polymorphism and the two main genetic variants have different drug binding properties. The binding characteristics of imatinib to AGP genetic variants and the possibility of its binding interactions were investigated by various methods. The results proved that binding of imatinib to the two main genetic variants is very different, the high affinity binding belongs dominantly to the F1-S variant. This interaction is accompanied with specific spectral changes (induced circular dichroism, UV change, intrinsic fluorescence quenching), suggesting that the bound ligand has chiral conformation that would largely overlap with other ligands inside the protein cavity. Binding parameters of Ka=1.7(+/-0.2)x10(6)M(-1) and n=0.94 could be determined for the binding on the F1-S variant at 37 degrees . Imatinib binding on the A variant is weaker and less specific. The binding affinity of imatinib to human serum albumin (nKa approximately 3 x 10(4)M(-1)) is low. Pharmacologically relevant binding interactions with other drugs can be expected on the F1-S variant of AGP.     

Selective binding of coumarin enantiomers to human alpha1-acid glycoprotein genetic variants

Coumarin-type anticoagulants, warfarin, phenprocoumon and acenocoumarol, were tested for their stereoselective binding to the human orosomucoid (ORM; AGP) genetic variants ORM 1 and ORM 2. Direct binding studies with racemic ligands were carried out by the ultrafiltration method; the concentrations of free enantiomers were determined by capillary electrophoresis. The binding of pure enantiomers was investigated with quinaldine red fluorescence displacement measurements. Our results demonstrated that all investigated compounds bind stronger to ORM 1 variant than to ORM 2. ORM 1 and human native AGP preferred the binding of (S)-enantiomers of warfarin and acenocoumarol, while no enantioselectivity was observed in phenprocoumon binding. Acenocoumarol possessed the highest enantioselectivity in AGP binding due to the weak binding of its (R)-enantiomer. Furthermore, a new homology model of AGP was built and the models of ORM 1 and ORM 2 suggested that difference in binding to AGP genetic variants is caused by steric factors.     

Structure of human alpha1-acid glycoprotein and its high-affinity binding site

Secondary and tertiary structures of human blood alpha(1)-acid glycoprotein, a member of the lipocalin family, have been studied for the first time by infrared and Raman spectroscopies. Vibrational spectroscopy confirmed details of the secondary structure and the structure content predicted by homology modeling of the protein moiety, i.e., 15% alpha-helices, 41% beta-sheets, 12% beta-turns, 8% bands, and 24% unordered structure at pH 7.4. Our model shows that the protein folds as a highly symmetrical all-beta protein dominated by a single eight-stranded antiparallel beta-sheet. Thermal dynamics in the range 20-70 degrees C followed by Raman spectroscopy and analyzed by principle component analysis revealed full reversibility of the protein motion upon heating dominated by decreasing of beta-sheets. Raman difference spectroscopy confirmed the proximity of Trp(122) to progesterone binding.     

Selective plasma protein binding of antimalarial drugs to alpha1-acid glycoprotein

Human plasma protein binding of six antimalarial agents of quinoline and acridine types was investigated by using spectroscopic techniques, affinity chromatography, ultrafiltration and HPLC methods. Induced circular dichroism (ICD) spectra showed binding of amodiaquine (AMQ), primaquine (PRQ), tafenoquine (TFQ), and quinacrine (QR) to alpha(1)-acid glycoprotein (AAG), the serum level of which greatly increases in Plasmodium infections. Association constant (K(a)) values of about 10(5)-10(6) M(-1) could be determined. Analysis of the ICD and UV spectra of the drug-AAG complexes suggested the inclusion of the ligands into the central hydrophobic cavity of the protein. Using the purified forms of the two main genetic variants of AAG, ICD data indicated the selective binding of AMQ and PRQ to the 'F1/S', while QR to the 'A' variant. Results of fluorescence experiments supported the AAG binding of these drugs and provided further insights into the binding details of TFQ and QR. Fluorescence and CD displacement experiments showed the high-affinity AAG binding of mefloquine (K(a) approximately 10(6) M(-1)). For this drug, inverse binding stereoselectivities were found with the 'F1/S' and 'A' genetic variants of AAG. HSA association constants estimated from affinity chromatography results lag behind (10(3)-10(5) M(-1)) the similar values derived for AAG. In case of chloroquine, no significant binding interaction was found either with AAG or HSA. Pharmacological aspects of the results are discussed.     

Topography of human plasma alpha1-acid glycoprotein.

Human plasma alpha1-acid glycoprotein, whose linear amino acid sequence has recently been elucidated (Schmid et al. (1973), Biochemistry 12, 2711), was further investigated with regard to its topography. Nitration of this protein and subsequent elucidation of the structures of the peptides containing modified tyrosine indicated that residues 27, 37, 78, 115, 127, and 157 are free, 50 and 91 are in an intermediate state, and 65, 74, 110, and 142 are buried. CD measurements between pH 10 and 12 demonstrated that the buried tyrosines are strongly hydrogen bonded and are probably responsible to a considerable extent for the stability of this protein. Of the three tryptophans of this protein, residue 122 proved to be partially reactive with Koshland reagent while the other two (25 and 160) were found to be unreactive. The state of the two disulfide bonds, established by differential reduction and alkylation with specific reagents, was shown to be of an intermediate type. Using carboxymethylation with bromoacetate at pH 7.0 for 8 days, the three histidines (97, 100, and 171) and methionine 111 could be shown to be in intermediate states. All lysines were treated with trinitrobenzenesulfonate and thus were assumed to be free. Of the 40 carboxylic groups, which were amidated with glycine methyl ester, 32 including the 14 sialyl residues were found to be free, six in an intermediate and the remaining two in a buried state. The present study describes the states of almost half of the amino acid residues of alpha1-acid glycoprotein, a knowledge important for the construction of a preliminary three-dimensional model of this conjugated protein.     

A three-step purification of human alpha 1-acid glycoprotein

alpha 1-Acid glycoprotein (AGP) was purified to homogeneity by a 3-step procedure using pseudo-ligand affinity chromatography on immobilized Cibacron blue F3GA, Procion red HE3B, and preparative column isoelectric focusing. The overall yield of the combined techniques was 88%. Analysis of the purified AGP by lectin affinity chromatography on immobilized Con A and immunoaffino-electrophoresis indicated that the most acidic form did not interact with the lectin, while the two more basic fractions possessed different affinities for Con A. In addition, 3 different populations of AGP were clearly separated by Con A affinity chromatography.     

Conformation selectivity in the binding of diazepam and analogues to alpha1-acid glycoprotein

Diazepam, a 1,4-benzodiazepine lacking chiral centre, exists in an equimolar mixture of two chiral conformers. Induced circular dichroism spectra for the binding of diazepam and its 3,3-dimethyl substituted analogues to alpha1-acid glycoprotein (AGP) revealed that opposite to human serum albumin, AGP preferably binds the P-conformers. Accordingly, slightly favoured binding of (R)-enantiomers of 3-alkyl derivatives having P-conformation was found. In case of 3-acyloxy derivatives, however, AGP preferably binds the (S)-enantiomers. Studies with the separated genetic variants of AGP proved similar binding affinities, but markedly different conformation selectivities. For diazepam bound by the F1-S variant, a P/M selectivity of about 2 could be estimated.     

Crystallization of alpha 1-acid glycoprotein

A possible link between cellular cyclic AMP content and Na+K+ATPase activity was investigated in homogenates of rat kidney. Enzyme kinetics of Mg2+ and Na+K+ATPase were run in the presence of cyclic AMP, dibutyryl cAMP and compounds expected to elevate cyclic AMP levels such as forskolin, a potent adenylate cyclase activator, IBMX, an inhibitor of phosphodiesterases, and the beta-agonist isoproterenol. Medullary Na+K+ATPase is strongly inhibited by cyclic AMP whereas cortical Na+K+ATPase was stimulated in the same conditions. The correlation between ATPase activity and cellular cyclic AMP content supports the concept of a possible regulation of the enzyme by cyclic AMP.     

Interaction of propafenone enantiomers with human alpha 1-acid glycoprotein

The interaction of propafenone enantiomers with human alpha 1-acid glycoprotein was studied using high-performance liquid chromatography. Each of the two optical antipodes interacted with one class of high-affinity binding sites characterized by Ka(R) = (6.18 +/- 0.93) x 10(5) M-1, n(R) = 1.34 +/- 0.09 for the (R)-isomer and Ka(S) = (8.93 +/- 1.82) x 10(5) M-1, n(S) = 0.99 +/- 0.08 for the (S)-isomer. Nonspecific binding to secondary low-affinity high-capacity binding site(s) was only slightly greater in the case of the (S)-enantiomer (n'k'(S) = (1.06 +/- 0.09) x 10(4) M-1) compared to the (R)-enantiomer (n'k'(R) = (6.87 +/- 0.72) x 10(3) M-1). It was concluded that both enantiomers interact with common single class of high-affinity binding sites on AAG (along with nonspecific binding) exhibiting only slight stereoselectivity for propafenone.     

Sequence comparison of human plasma alpha1-acid glycoprotein and the immunoglobulins

The amino acid sequence of human plasma alpha1-acid glycoprotein, upon comparison with the sequences of other blood proteins, was shown to possess significant similarity with the immunoglobulins. Employing direct and corrected sequence identity, the average mutation value and two different computer comparisons for the evaluation of sequence similarity, the following two regions of this alpha-globulin, which account for approximately half of the total amino acid sequence of the protein, were found to possess sequence similarity with the immunoglobulins. a) The region from residues 77 through 125 proved to be related to the variable region of several human H and L chains, and b) the region from residues 136 through 166 was found to be related not only to the constant region of a human and a mouse L chain but also to the third and fourth constant region of a rabbit and a human H chain, respectively. These results suggest that alpha1-acid glycoprotein is probably related to the immunoglobulins and further suggest that it possibly diverged from the immunoglobulin evolutionary tree prior to the formation of the primitive L chain.     

Biliverdin is the endogenous ligand of human serum alpha1-acid glycoprotein

α₁-Acid glycoprotein (AAG), an acute phase component of the human serum, is a prominent member of the lipocalin family of proteins showing inflammatory/immunomodulatory activities and promiscuous drug binding properties. Both three-dimensional structure of AAG and its precise biological function are still unknown and only a few endogenous AAG ligands have been described to date. CD spectroscopic studies performed with commercial AAG and the separated genetic variants revealed high-affinity binding of biliverdin (BV) and biliverdin dimethyl ester to the ‘F1/S’ fraction of the protein. The preferential accommodation of the right-handed, P-helicity conformers of the pigments by the protein matrix resulted in strong induced CD activity, which was utilized for estimation of the binding parameters and to locate the binding site. It was concluded that both pigments are bound in the central β-barrel cavity of AAG, held principally by hydrophobic interactions. Possible biological implications of the BV binding ability of AAG with special emphasis on the heme oxygenase-1 pathway are discussed.     

Function-stabilizing mechanism of plasminogen activator inhibitor type 1 induced upon binding to alpha1-acid glycoprotein

Recently, we found that alpha(1)-acid glycoprotein (AGP), one of the major acute-phase proteins, forms a function-stabilizing complex with plasminogen activator inhibitor 1 (PAI-1). In this study we describe the mechanism by which AGP, as well as its recombinant fragment AGP(118)(-)(201), interacts exclusively with the active form of PAI-1 and stabilizes its conformation. The binding domain of PAI-1 for AGP was initially mapped by antibodies reacting with the well-defined PAI-1 epitopes and then verified in binding assays utilizing a library of PAI-1 mutants. The latter consisted of PAI-1 molecules with individual, tandem, or grouped mutations in the epitope region of MA-55F4C12, MA-33B8, MA-33H1F7, MA-44E4, and MA-8H9D4. Solid-phase binding experiments showed that only MA-8H9D4 did not bind to the PAI-1/AGP complex, indicating that its epitope is hidden upon binding of PAI-1 to AGP. Consistently, only PAI-1 mutants with substitutions in the region of R300-D305, constituting the MA-8H9D4 epitope, showed a lack of binding or severe deficit in both the capacity and affinity of binding to AGP. These results support a location of the binding site close to the epitope within the segment connecting the regions hI with S5A. In conclusion, our present data suggest that AGP binding stabilizes the active conformation of PAI-1 by restricting the movement of beta-sheet A and thereby preventing insertion of the reactive center loop.     

One-step isolation of alpha 1-acid glycoprotein.

alpha 1-Acid glycoprotein could be isolated by a one-step extraction method from human sera and plasma. Protein recovered in the water phase after extraction with phenol at 70 degrees C for 20 min was verified as human alpha 1-acid glycoprotein when it was compared with the reference standard human alpha 1-acid glycoprotein by Ouchterlony double immunodiffusion, sodium dodecylsulfate-polyacrylamide gel electrophoresis, Western blot analysis, and periodic acid-Schiff stain. The present isolation procedure is simple and fast, and can extract about 81% of the total alpha 1-acid glycoprotein in the sera and plasma, as determined by radial immunodiffusion.     

Evolution of human alpha 1-acid glycoprotein genes and surrounding Alu repeats

There is a mosaic pattern of variation between the two tandemly arranged human alpha 1-acid glycoprotein genes. Both the synonymous and the nonsynonymous sites of exons 3 and 4 are more divergent than the rest of the gene, suggesting that they have had a different evolutionary history. Comparisons of the two gene sequences with rat AGP indicate that exons 3 and 4 of AGP2 have been evolving without functional constraint since their divergence from AGP1. It is proposed that the conserved region of the gene has been homogenized recently by gene conversion with the homologous regions of AGP1. The Alu sequences surrounding the genes appear to have been involved in both the gene duplication and the gene conversion events.     

Association studies of aggregated aqueous lutein diphosphate with human serum albumin and alpha1-acid glycoprotein in vitro: evidence from circular dichroism and electronic absorption spectroscopy

Water-dispersible C40 carotenoid derivatives, with increased utility in mammalian therapeutic applications, include natural stereoisomer-based (3R,3'R,6'R)-lutein (beta,epsilon-carotene-3,3'-diol) derivatives. Esterification with inorganic phosphate and conversion to the sodium salt produced compounds (lutein diphosphate sodium salt; 'LdP') capable of forming red-orange aqueous suspensions after addition to USP-purified water. The aqueous dispersibility of this diphosphate salt reached 29 mg/mL without the addition of heat, detergents, co-solvents, or other additives, and was a potent direct scavenger of superoxide anion (by EPR spectroscopy) in an isolated human neutrophil assay. In the current study, preliminary evidence of the aqueous aggregation of this compound in EPR studies was confirmed using circular dichroism (CD) and electronic absorption (UV-vis) spectroscopy. Evidence for H-type ('card-pack') and J-type ('head-to-tail') self-assemblies was obtained. In vitro analysis of the potential binding interaction between LdP and human serum albumin (HSA) and alpha1-acid glycoprotein (AGP) revealed only non-specific binding with HSA (and none with AGP), contrasting with previous reports of direct interaction between astaxanthin-based soft drugs and the major plasma protein albumin. The rapid in vivo cleavage of this phosphodiester by promiscuous mammalian phosphatases may overcome the aqueous aggregation of the formulated compound. This difference in potential plasma protein interaction with prior reports reflects the subtle structural differences inherent in either the parent carotenoid scaffolds and/or the esterifying moieties.     

Immobilization of alpha(1)-acid glycoprotein for chromatographic studies of drug-protein binding

A new method for preparing immobilized alpha1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M(-1), respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.