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Background: Natural mutations of R218 in human serum albumin (HSA) result in an increased affinity for L-thyroxine and lead to the autosomal dominant condition of familial dysalbuminemic hyperthyroxinemia., Methods: Binding was studied by equilibrium dialysis and computer modeling., Results: Ten of 32 other isoforms tested had modified high-affinity hormone binding. L-thyroxine has been reported to bind to four sites (Tr) in HSA; Tr1 and Tr4 are placed in the N-terminal and C-terminal part of the protein, respectively. Site-directed mutagenesis gave new information about all the sites., Conclusions: It is widely assumed that Tr1 is the primary hormone site, and that this site, on a modified form, is responsible for the above syndrome, but the binding experiments with the genetic variants and displacement studies with marker ligands indicated that the primary site is Tr4. This new assignment of the high-affinity site was strongly supported by results of MM-PBSA analyses and by molecular docking performed on relaxed protein structure. However, dockings also revealed that mutating R218 for a smaller amino acid increases the affinity of Tr1 to such an extent that it can become the high-affinity site., General Significance: Placing the high-affinity binding site (Tr4) and the one which can result in familial dysalbuminemic hyperthyroxinemia (Tr1) in two very different parts of HSA is not trivial, because in this way persons with and without the syndrome can have different types of interactions, and thereby complications, when given albumin-bound drugs. The molecular information is also useful when designing drugs based on L-thyroxine analogues., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products., Methods: Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration., Results: N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present., Conclusion: The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations., General Significance: N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: A strategy for preventing cisplatin nephrotoxicity due to enhanced oxidative stress and inflammatory response is highly desirable. Thioredoxin-1 (Trx), an endogenous redox-active protein, has a short retention time in the blood. A long acting form of Trx, human serum albumin-Trx (HSA-Trx), was produced by recombinant HSA fusion and its effectiveness in preventing cisplatin nephrotoxicity was examined., Methods: HSA-Trx was prepared in Pichia expression system. Cisplatin-induced nephropathy mouse model was established by a single administration of cisplatin., Results: Compared to saline, Trx or N-acetylcysteine, an intravenous administration of HSA-Trx attenuated the cisplatin-induced elevation in serum creatinine, blood urea nitrogen and urinary N-acetyl-β-d-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx caused a substantial reduction in the histological features of renal tubular injuries and the apoptosis-positive tubular cells. Changes in superoxide, 8-OHdG, glutathione and nitrotyrosine levels indicated that HSA-Trx significantly suppressed renal oxidative stress. HSA-Trx also suppressed the elevation of TNF-α, IL-1β and IL-6. Administered fluorescein isothiocyanate-labeled HSA-Trx was found partially localized in the proximal tubular cells whereas majority remained in the blood circulation. Specific cellular uptake and the scavenging of intracellular reactive oxygen species by HSA-Trx were observed in HK-2 cells., Conclusion: HSA-Trx could be a novel and effective approach for preventing cisplatin nephrotoxicity due to its prolonged anti-oxidative and anti-inflammatory action not only in extracellular compartment but also inside the proximal tubular cell., General Significance: We report the renoprotective effect of HSA-Trx against cisplatin nephrotoxicity. This work would enhance developing therapeutics against acute kidney injuries including cisplatin nephrotoxicity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Human serum albumin acts as a reservoir and transport protein for endogenous (e.g. fatty acids or bilirubin) and exogenous compounds (e.g. drugs or nutrients) in the blood. The binding of a drug to albumin is a major determinant of its pharmacokinetic and pharmacodynamic profile., Scope of Review: The present review discusses recent findings regarding the nature of drug binding sites, drug-albumin binding in certain diseased states or in the presence of coadministered drugs, and the potential of utilizing albumin-drug interactions in clinical applications., Major Conclusions: Drug-albumin interactions appear to predominantly occur at one or two specific binding sites. The nature of these drug binding sites has been fundamentally investigated as to location, size, charge, hydrophobicity or changes that can occur under conditions such as the content of the endogenous substances in question. Such findings can be useful tools for the analysis of drug-drug interactions or protein binding in diseased states. A change in protein binding is not always a problem in terms of drug therapy, but it can be used to enhance the efficacy of therapeutic agents or to enhance the accumulation of radiopharmaceuticals to targets for diagnostic purposes. Furthermore, several extracorporeal dialysis procedures using albumin-containing dialysates have proven to be an effective tool for removing endogenous toxins or overdosed drugs from patients., General Significance: Recent findings related to albumin-drug interactions as described in this review are useful for providing safer and efficient therapies and diagnoses in clinical settings. This article is part of a Special Issue entitled Serum Albumin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Oxidative damage results in protein modification, and is observed in numerous diseases. Human serum albumin (HSA), the most abundant circulating protein in the plasma, exerts important antioxidant activities against oxidative damage., Scope of Review: The present review focuses on the characterization of chemical changes in HSA that are induced by oxidative damage, their relevance to human pathology and the most recent advances in clinical applications., Major Conclusions: The antioxidant properties of HSA are largely dependent on Cys34 and its contribution to the maintenance of intravascular homeostasis, including protecting the vascular endothelium under disease conditions related to oxidative stress. Recent studies also evaluated the susceptibility of other important amino acid residues to free radicals. The findings suggest that a redox change in HSA is related to the oxidation of several amino acid residues by different oxidants. Further, Cys34 adducts, such as S-nitrosylated and S-guanylated forms also play an important role in clinical applications. On the other hand, the ratio of the oxidized form to the normal form of albumin (HMA/HNA), which is a function of the redox states of Cys34, could serve as a useful marker for evaluating systemic redox states, which would be useful for the evaluation of disease progression and therapeutic efficacy., General Significance: This review provides new insights into our current understanding of the mechanism of HSA oxidation, based on in vitro and in vivo studies. This article is part of a Special Issue entitled Serum Albumin., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: 4Z,15Z-bilirubin-IXα (BR), an endogenous toxic compound that is sparingly soluble in water, binds human serum albumin (HSA) with high affinity in a flexible manner. Our previous findings suggest that both Lys195 and Lys199 in subdomain IIA are important for the high-affinity binding of BR, and especially Lys199 in stand-alone domain II plays a prominent role in the renal elimination of BR. Our hypothesis is that HSA-domain II with high BR binding would be a useful therapeutic agent to treat hyperbilirubinemia in patients with impaired liver function., Methods: Unbound BR concentrations were determined using a modified HRP assay. To evaluate the effect of pan3_3-13 domain II mutant in promoting urinary BR excretion, the serum concentration and urinary excretion amount of BR were determined using bile duct ligation mice., Results: After three or six rounds of panning, pan3_3-13 and pan6_4 were found to have a significantly higher affinity for BR than wild-type domain II. Administration of pan3_3-13 significantly reduced serum BR level and increased its urinary excretion in the disease model mice as compared to wild-type domain II treatment., Conclusions: These results suggest that pan3_3-13 has great potential as a therapeutic agent that promotes urinary BR excretion in hyperbilirubinemia., General Significance: This is the first study to be applied to other HSA bound toxic compounds that are responsible for the progression of disease, thereby paving the way for the development of non-invasive and cost effective blood purification treatment methods., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Human serum albumin with modified plasma half-life will be useful for clinical purposes. Therefore, the pharmacokinetics of three of each of the following types of genetic variants, and of their corresponding normal albumin, were examined in mice: N-terminally elongated, C-terminally truncated and glycosylated albumins. Isoforms differing from the normal protein by three or more amino acids, especially two of the truncated forms, had shorter half-lives. The effect of glycosylation depended on the position of attachment: in domain II it increased half-life, whereas in domain I and III it had no significant effect. Liver, kidney and spleen uptake clearances were also modified. The pronounced changes in half-life of the two truncated variants and the glycosylated isoform could be explained, at least partly, by large changes in organ uptakes; in the remaining six cases, different effects were registered. Such information should be useful when designing therapeutical albumin products for, e.g., drug delivery systems. In addition to various types of cell endocytosis, leading to intracellular destruction or recycling of the proteins, the metabolism of the alloalbumins could be affected by plasma enzymes. No correlation was found between mutation-induced changes in the pharmacokinetic parameters and changes in alpha-helical content or changes in heat stability as represented by DeltaH(v).

The pharmacokinetics of 17 genetic variants of human serum albumin with single-residue mutations and their corresponding normal albumin were studied in mice. In all cases, the plasma half-life was affected, but only variants with +2 changes in charge prolonged it, whereas changes in hydrophobicity decreased it. Good positive and negative correlations were found between changes in alpha-helical content taking place in domains I+III and domain II, respectively, and changes in half-lives. No correlation was found to type of mutation or to changes in heat stability as represented by DeltaH(v). Liver and kidney uptake clearances were also modified: alpha-helical changes of domains I+III showed good negative correlations to both types of clearances, whereas changes in domain II only had a good positive correlation to kidney uptake clearance. No correlation between the other molecular changes and organ uptakes was observed. The relatively few correlations between changes in molecular characteristics and the organ uptakes of the variants are most probably due to different handling by plasma enzyme(s) and the various types of cell endocytosis. Of the latter, most lead to destruction of albumin, but at least one results in recycling of the protein. The present information should be useful when designing recombinant, therapeutical albumins or albumin products with a modified plasma half-life.

The cellular interaction of proteins modified with advanced glycation end-products (AGEs) is believed to induce several different biological responses, which are involved in the development of diabetic vascular complications. We report here that the ratio of protein glycation is implicated in its ligand activity to scavenger receptors. Although highly-modified AGE-bovine serum albumin (high-AGE-BSA) was significantly recognized by human monocyte-derived macrophages and Chinese hamster ovary cells which overexpress such scavenger receptors as CD36, SR-BI (scavenger receptor class B type-I), and LOX-1 (Lectin-like Ox-LDL receptor-1), the mildly-modified-AGE-BSA (mild-AGE-BSA) did not show any ligand activity to these cells. Furthermore, when (111)In-labeled high- or mild-AGE-BSA were injected into the tail vein of mice, the high-AGE-BSA was rapidly cleared from the circulation whereas the clearance rate of the mild-AGE-BSA was very slow, similar to the native BSA. These results demonstrate the first evidence that the ligand activity of the AGE-proteins to the scavenger receptors and its pharmacokinetic properties depend on their rate of modification by AGEs, and we should carefully prepare the AGE-proteins in vitro to clarify the physiological significance of the interaction between the AGE-receptors and AGE-proteins.

The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs. The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase in carbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in alpha-helical content as the result of oxidation was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in the pharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A and R410A mutants were affected (extent of effect: R410A > K199A > H146A). The liver clearance of these proteins is closely correlated to hydrophobicity (r = 0.929, P < 0.01) and the net charge of the proteins (r=0.930, P < 0.01). The rate of elimination of HSA is closely related to the hydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.

The binding of flunitrazepam (FNZP) by human alpha1-acid glycoprotein (hAGP) and the relationships between the extent of drug binding and desialylation and the genetic variants of hAGP were examined. The photolabeling specificity of [3H]FNZP was confirmed by findings in which other hAGP-binding ligands inhibited the formation of covalent bonds between [3H]FNZP and hAGP. The photolabeling of asialo-hAGP suggested that sialic acid does not involve in the binding of [3H]FNZP. No difference in the labeling could be found between the F1*S variants and A variant. Similarly, FNZP did not show a difference in binding affinity to the two genetic variants of hAGP. Sequence analysis of the photolabeled peptide indicated a sequence corresponding to Tyr91-Arg105 of hAGP.

Reversible thermal denaturation of 33 genetic variants of human serum albumin (HSA) appeared to be a two-state process when studied by circular dichroism (CD). Fourteen single-residue variants have Tm values (midpoint of denaturation) higher than, and nine have Tm values lower than, their endogenous, wild-type counterpart. Nine single-residue variants have DeltaHv values (van't Hoff enthalpy) higher than, and 14 have DeltaHv values lower than, normal albumin. All types of combinations of positive and negative DeltaTm values and Delta(DeltaHv) values were found. Good linear correlations between mutation-induced changes of alpha-helical content and Delta(DeltaHv) values, but not DeltaTm values, were found especially for the variants mutated in domains I and III. The effect of altered chain length and glycosylation on Tm and DeltaHv was also studied. For all variants, no clear relationship was found between the changes in the thermodynamic parameters and the type of substitution, changes in protein charge or hydrophobicity. However, the protein changes taking place in domain I have a rather uniform effect (almost all of the nine variants have positive DeltaTm values and negative Delta(DeltaHv) values, i.e., they denature more easily than normal albumin but they do so at a higher temperature). The present results can be of both protein chemical relevance and of clinical interest, because they could be useful when designing stable, recombinant HSAs for clinical applications.

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.

The recombinant human serum albumin (rHSA) dimer, which was cross-linked by a thiol group of Cys-34 with 1,6-bis(maleimido)hexane, has been physicochemically characterized. Reduction of the inert mixed-disulfide of Cys-34 beforehand improved the efficiency of the cross-linking reaction. The purified dimer showed a double mass and absorption coefficient, but unaltered molar ellipticity, isoelectric point (pI: 4.8) and denaturing temperature (65 degrees C). The concentration dependence of the colloid osmotic pressure (COP) demonstrated that the 8.5 g dL(-1) dimer solution has the same COP with the physiological 5 g dL(-1) rHSA. The antigenic epitopes of the albumin units are preserved after bridging the Cys-34, and the circulation lifetime of the 125I-labeled variant in rat was 18 h. A total of 16 molecules of the tetrakis[(1-methylcyclohexanamido)phenyl]porphinatoiron(II) derivative (FecycP) is incorporated into the hydrophobic cavities of the HSA dimer, giving an albumin-heme hybrid in dimeric form. It can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) like hemoglobin or myoglobin. Magnetic circular dichroism (CD) revealed the formation of an O2-adduct complex and laser flash photolysis experiments showed the three-component kinetics of the O2-recombination reaction. The O2-binding affinity and the O2-association and -dissociation rate constants of this synthetic hemoprotein have also been evaluated.

The capability of octanoate, N-acetyl-L-tryptophanate (N-AcTrp) and other ions of fatty acids and amino acids to stabilize human serum albumin (HSA) against thermal and oxidative stress was studied. Native-PAGE showed that octanoate, and more hydrophobic fatty acids anions, stabilizes the monomeric form of HSA during heating at 60 degrees C for 30 min. Heating in the presence of octanoate did not change the far-UV CD-spectrum. The stabilizing role of octanoate also showed as an increase in denaturation temperature and calorimetric enthalpy, determined by differential scanning calorimetry (DSC). N-AcTrp, which was found to compete with octanoate for a common high-affinity site, has only a minor stabilizing effect. By contrast, no effect was found for l-tryptophanate or N-acetyl-L-cysteinate. Any ligand effect on oxidation was examined by using 2,2' -azobis(2-amidino-propane)dihydrochloride (AAPH) as oxidizing agent. One hour of incubation resulted in the formation of the same number of carbonyl groups, whether octanoate or one of the above mentioned amino acids was present or not. However, the number of groups formed after 24 h of incubation was significantly decreased in the presence of L-tryptophanate and, especially, N-AcTrp. The effect of 1-h incubation with AAPH on the oxidative status of 34-Cys was studied by the HPLC technique. It was found that N-AcTrp, but not octanoate, has a large protecting effect on the sulfhydryl group. Thus, octanoate has the greatest stabilizing effect against heat, whereas the presence of N-AcTrp diminishes oxidation of HSA.

Recombinant wild-type human serum albumin (rHSA), the single-residue mutants K199A, K439A and K525A and the triple-residue mutant K199A/K439A/K525A were produced using a yeast expression system. Portions of the rHSA were glycated to different degrees (2.5-250 mM D-glucose). As detected by far-UV and near-UV CD, intrinsic tryptophan-fluorescence and probed by 1,1'-bis(4-anilino)naphthalene-5,5-disulfonic acid, the single-residue mutations had no effect on albumin conformation, whereas the triple-residue mutation and glycation caused conformational changes. The triple-residue mutation and glycation had comparable increased effects on high-affinity binding of warfarin (site I), but decreased effects on high-affinity binding of dansylsarcosine (site II) and the esterase-like activity of albumin. The relation between plasma half-lives in rats were found to be glycated rHSA (50 mM glucose)

Alpha(1)-acid glycoprotein (AGP) is a glycoprotein that consists of 183 amino acid residues and five carbohydrate chains and binds to neutral and basic drugs. We examined the structural properties and ligand-binding capacity of AGP in interactions with reverse micelles. Also, detailed information was obtained by comparing several different states of AGP. Interaction with reverse micelles induced a unique conformational transition (beta-sheet to alpha-helices) in AGP and decreased the binding capacity for the basic drug, chlorpromazine and the steroid hormone, progesterone to AGP. These structural conformations are very similar to those observed under conditions of acidity and high ionic strength (pH 2.0, 1.5 M NaCl). This structure seems to be an intermediate between the native state and the denatured state, possibly a molten globule. The present results suggest that when AGP interacts with the biomembrane, it undergoes a structural transition to a unique structure that differs from the native and denatured states and has a reduced ligand-binding capacity.

Benzodiazepine (BDZ) is generally thought to bind to site II of human serum albumin (HSA), also known as the indole-BDZ site, which is located at subdomain III A of the molecule. However, differences in the binding characteristics of BDZ drugs with HSA have been reported. The photolabeling profiles of HSA with [(3)H]flunitrazepam (FNZP) in the presence and absence of diazepam (DZP) were shown to be identical, suggesting that each drug primarily binds to different regions. The results of fluorescent probe displacement experiments showed that FNZP failed to decrease the fluorescence of dansylsarcosine to an extent similar to that of DZP. In the photoinhibition experiment, site I and site II ligands failed to inhibit the photoincorporation of [(3)H]FNZP to HSA. In order to evaluate the photolabeling specificity of FNZP, an attempt was made to photolabel alpha(1)-acid glycoprotein (AGP) which also binds BDZ with similar affinity as HSA. The effect of myristate (MYR) and DZP on the FNZP photolabeling of these two major drug binding plasma proteins was examined. Photoincorporation was inhibited when HSA was photolabeled with [(3)H]FNZP in the presence of MYR but not in the presence of DZP. Conversely, DZP inhibited the photolabeling of [(3)H]FNZP to AGP. These results suggest that FNZP interacts with HSA at regions which are not located in the preformed binding pocket of subdomain III A.

It is well known that the subdomain III A (site II) of human serum albumin (HSA) binds a variety of endogenous and exogenous substances. However, the nature of the microenvironment of the binding site remains unclear. Ketoprofen (KP), an arylpropionic acid NSAID which contains a benzophenone moiety, was used as a photoaffinity labeling agent to label the binding region. Subsequent CNBr cleavage of the photolabeled HSA revealed that the 11.6 kDa and 9.4 kDa fragments contained most of the incorporated radioactivity. Competition experiments showed that the 11.6 kDa fragment contains the common binding region for site II ligands. This fragment was redigested with Achromobacter lyticus protease I (AP-I) and the amino acid sequence of the photolabeled peptide was determined to be XCTESLVNRR, which corresponds to the sequence 476C-485K of HSA. The complete amino acid sequence of the corresponding AP-I digested HSA peptide encompasses residues 476 to 499, which form helices 5 and 6 of subdomain III A. The HSA-Myr X-ray crystallography data showed that helix 5 is involved to the least extent in ligand binding. A docking model provided further support that helix 6 represents the photolabeled region of KP.

The relationship between the two principal ligand binding sites, sites I and II, on human serum albumin (HSA) was quantitatively and qualitatively examined by equilibrium dialysis and fluorescence spectroscopy. Among the three subsite markers to site I, only the binding of dansyl-L-asparagine (DNSA), which is a subsite Ib marker (K. Yamasaki et al., Biochim. Biophys. Acta 1295 (1996) 147), was inhibited by the simultaneous binding of a site II ligand, such as ibuprofen and diazepam. This indicates that, in contrast to subsite Ib, subsites Ia and Ic do not strongly interact with site II. The thermodynamic characteristics for the coupling reaction between DNSA and ibuprofen and between DNSA and diazepam, which gave positive coupling free energies and negative values for both coupling enthalpy and entropy, indicated that the reaction process was entropically driven. Increase of pH from 6.5 to 8.2 caused an increase in coupling constant and entropy for the mutual antagonism between DNSA and the site II ligands on binding to HSA. The site II ligand-induced red-shift of lambda(max) and solvent accessibility of DNSA in subsite Ib were decreased when the albumin molecule was isomerized from the neutral (N) to the base (B) conformation in the physiological pH region. Based on these findings, we conclude that a 'competitive' like strong allosteric regulation exists for the binding of these two ligands to the N conformer, whereas for the B conformer this interaction can be classified as nearly 'independent'. Since the distance between Trp-214, which resides within the site I subdomain, and Tyr-411, which is involved in site II, is increased by 6 A during the N-B transition (N.G. Hagag et al., Fed. Proc. 41 (1982) 1189), we propose a mechanism for the pH-dependent antagonistic binding between subsite Ib and site II, which involves the transmission of ligand-induced allosteric effects from one site to another site, modified by changes in the spatial relationship of sites I and II caused by the N-B transition.

The microenvironment surrounding Cys-34 in human serum albumin (HSA) has been studied using acrylodan, a cysteine-specific fluorescence probe. Fluorescence emission maximum (Em(max)) of acrylodan covalently attached to Cys-34 was observed at 476 nm, which was nearly the same as for acetonitrile. The Em(max) exhibited a shift toward longer wavelength with oleate binding. The acrylodan fluorescence of HSA exhibited heterogeneous decay kinetics, which adequately fit a double-exponential decay model. When three or more oleate molecules were bound to one albumin molecule, the fractional intensity was shifted in favor of the shorter lived component. These results suggest that oleate binding induces a conformational or dynamic change which is localized in the Cys-34 region. Unfolding studies with guanidine-HCl indicate that Cys-34 appears to be located on the surface of HSA molecule and that it is protected by adjacent amino acid residues. Solvent accessibility of acrylodan with HSA in the absence and presence of oleate was determined from acrylamide quenching, and suggests that oleate binding enhances the solvent exposure of the acrylodan fluorophore. In order to determine the nature of the electrostatic potential near Cys-34, the quenching rate constants for anionic (iodide) and cationic (thallium) quenchers were determined as a function of ionic strength of solvent. The ionic strength dependence of quenching indicated that there was an electrostatic attractive force between the fluorophore and both ionic quenchers. These results are consistent with a model in which amphoteric charges which arise from charged amino acid residues were surrounding Cys-34. Interestingly, oleate binding resulted in changes in the spatial relationships between acrylodan and these charged residues. Thus, the experiments described herein provide the information concerning an oleate-induced alteration in the nature of the local environment surrounding Cys-34 and suggests that long chain fatty acid binding provides a method for regulating the radical-trapping antioxidant activity of Cys-34 in HSA in vivo.

Human serum albumin (HSA) possesses at least three sites or areas for high-affinity binding of drugs. Of these sites, site I was investigated by series of ultrafiltration and equilibrium dialysis experiments. Three ligands, acenocoumarol, dansyl-L-asparagine (DNSA) and n-butyl p-aminobenzoate (n-butyl p-ABE) were employed as marker ligands. Each ligand binds to a single high-affinity site on HSA, and binding studies with different pairs of the ligands revealed independent high-affinity binding. Preliminary displacement studies performed with the typical site I binding drugs warfarin, phenylbutazone and iodipamide showed different displacement patterns of the three marker ligands. These studies were followed by stringent competition experiments involving all possible combinations of the three test ligands themselves and of these and the three marker ligands. On the basis of the results obtained it seems that the acenocoumarol and DNSA binding regions correspond to the warfarin and azapropazone binding regions, respectively, of site I reported by others (Fehske, Schläfer, Wollert and Müller (1982) Mol. Pharmacol. 21, 387-393). The new binding region, represented by n-butyl p-ABE, is probably located adjacent to the acenocoumarol binding region but apart from that of DNSA. We have elaborated a model for binding site I in which we propose novel nomenclatures, region Ia, Ib, and Ic for the acenocoumarol, DNSA and n-butyl p-ABE binding regions, respectively. Furthermore, the relation between these regions and the high-affinity binding sites for other drugs have been discussed.

Carbonyl reductase from rabbit kidney was inactivated by phenylglyoxal (PGO) and 2,4,6-trinitrobenzenesulfonate sodium (TNBS). NADP+ protected the enzyme from the inactivations by PGO and TNBS, suggesting that essential arginine and lysine residues are located in coenzyme-binding domain of the enzyme. Judging from the effects of PGO-treated enzymes in the presence and in the absence of NADP+ on the fluorescence intensity of NADPH, one essential arginine residue in coenzyme-binding domain was found to have a role in the binding of NADPH to the enzyme. Indomethacin afforded a significant protection against inactivation of the enzyme by PGO, whereas it could not protect the enzyme from the inactivation by TNBS. It is reasonable to postulate that indomethacin interacts at least in part with or near one essential arginine residue in coenzyme-binding domain of carbonyl reductase from rabbit kidney.

The kinetic mechanism for the reduction of acetohexamide catalyzed by carbonyl reductase from rabbit kidney was investigated. The initial velocity and product inhibition studies indicated that the enzymatic reaction follows an ordered Bi Bi mechanism, in which NADPH binds to the enzyme first and NADP leaves last. This kinetic mechanism was confirmed on the basis of the dead-end inhibition by Cibacron Blue and the binding of NADPH and NADP to the free enzyme. However, whether or not coenzyme-induced isomerization is involved in the enzymatic reaction remains to be clarified. In kinetic studies of inhibition of the enzyme by therapeutically active drugs, indomethacin and befunolol were found to be noncompetitive and competitive inhibitors, respectively, with respect to acetohexamide.