Transfer of ANS-Like Drugs from Micellar Drug Delivery Systems to Albumin Is Highly Favorable and Protected from Competition with Surfactant by "Reserved" Binding Sites.

Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitate─a process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.

A surprisingly simple three-state generic process for reversible protein denaturation by trifluoroethanol.

Despite the rich knowledge of the influence of 2,2,2-trifluoroethanol (TFE) on the structure and conformation of peptides and proteins, the mode(s) of TFE-protein interactions and the mechanism by which TFE reversibly denatures a globular protein remain elusive. This study systematically examines TFE-induced equilibrium transition curves for six paradigmatic globular proteins by using basic fluorescence and circular dichroism measurements under neutral pH conditions. The results are remarkably simple. Low TFE invariably unfolds the tertiary structure of all proteins to produce the obligate intermediate (I) which retains nearly all of native-state secondary structure, but enables the formation of extra α-helices as the level of TFE is raised higher. Inspection of the transitions at once reveals that the tertiary structure unfolding is always a distinct process, necessitating the inclusion of at least one obligate intermediate in the TFE-induced protein denaturation. It appears that the intermediate in the minimal unfolding mechanism N⇌I⇌D somehow acquires higher α-helical propensity to generate α-helices in excess of that in the native state to produce the denatured state (D), also called the TFE state. The low TFE-populated intermediate I may be called a universal intermediate by virtue of its α-helical propensity. Contrary to many earlier suggestions, this study dismisses molten globule (MG)-like attribute of I or D.

Bioactive Properties of a Novel Antibacterial Dye Obtained from Laccase-Mediated Oxidation of 8-Anilino-1-naphthalenesulfonic Acid.

Fungal laccase obtained from a Cerrena unicolor strain was used as an effective biocatalyst for the transformation of 8-anilino-1-naphthalenesulfonic acid into a green-coloured antibacterial compound, which can be considered as both an antimicrobial agent and a textile dye, simultaneously. The process of biosynthesis was performed in buffered solutions containing methanol as a co-solvent, allowing better solubilisation of substrate. The transformation process was optimised in terms of the buffer pH value, laccase activity, and concentrations of the substrate and co-solvent. The crude product obtained exhibited low cytotoxicity, antibacterial properties against Staphylococcus aureus and Staphylococcus epidermidis, and antioxidant properties. Moreover, the synthesised green-coloured compound proved non-allergenic and demonstrated a high efficiency of dyeing wool fibres.

Interaction of cyclotide Kalata B1 protein with model cellular membranes of varied electrostatics.

A uni-molecular layer of lipids at air-water interface mimicking one of the leaflets of the cellular membrane provides a simple model to understand the interaction of any foreign molecules with the membrane. Here, the interactions of protein Kalata B1 (KB1) of cyclotide family with the phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG), and 1,2-distearoyl-sn-glycero-3-ethylphosphocholine chloride salt (DSEPC) have been investigated. The addition of KB1 induces a change in pressure of the lipid monolayers. The characteristic time of the change in pressure is found to be dependent on the electrostatic nature of the lipid. Even though the protein is weakly surface active, it is capable of modifying the phase behavior and elastic properties of lipid monolayers with differences in their strength and nature making the layers more floppy. The KB1-lipid interaction has been quantified by calculating the excess Gibb's free energy of interaction and the 1-anilino-8-naphthalenesulfonate (ANS) binding studies. The interaction with zwitterionic DPPC and negatively charged DPPG lipids are found to be thermodynamically favorable whereas the protein shows a weaker response to positively charged DSEPC lipid. Therefore, the long ranged electrostatic is the initial driving force for the KB1 to recognize and subsequently attach to a cellular membrane. Thereafter, the hydrophobic region of the protein may penetrate into the hydrophobic core of the membrane via specific amino acid residues.

Exploring the inhibitory effects of liquiritigenin against tau fibrillation and related neurotoxicity as a model of preventive care in Alzheimer's disease.

Aggregation of tau protein into the form of insoluble amyloid fibrils is linked with Alzheimer's disease. The identification of potential small molecules that can inhibit tau protein from undergoing aggregation has received a great deal of interest, recently. In the present study, the possible inhibitory effects of liquiritigenin as a member of chiral flavanone family on tau amyloid fibrils formation and their resulting neurotoxicity were assessed by different biophysical and cellular assays. The inhibitory effect of the liquiritigenin against tau amyloid formation was investigated using thioflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence spectroscopy, Congo red (CR) binding assays, transmission electron microscopy (TEM) analysis, and circular dichroism (CD) spectroscopy. Neurotoxicity assays were also performed against neuron-like cells (SH-SY5Y) using 3-(4,5-Dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS), catalase (CAT) and caspase-3 activity measurements. We found that liquiritigenin served as an efficient inhibitor of tau amyloid fibrils formation through prevention of structural transition in tau structure, exposure of hydrophobic patches and their associated neurotoxicity mediated by decrease in the production of ROS and caspase-3 activity and elevation of CAT activity. These data may finally find applications in the development of promising inhibitors against amyloid fibril formation and treatment of Alzheimer's disease.

Enhanced accessibility and hydrophobicity of amyloidogenic intermediates of the ß2-microglobulin D76N mutant revealed by high-pressure experiments.

ß2-Microglobulin (ß2m) is the causative protein of dialysis-related amyloidosis. Its unfolding mainly proceeds along the pathway of NC →UC ⇄ UT, whereas refolding follows the UT → IT (→NT) →NC pathway, in which N, I, and U are the native, intermediate, and unfolded states, respectively, with the Pro32 peptidyl-prolyl bond in cis or trans conformation as indicated by the subscript. It is noted that the IT state is a putative amyloidogenic precursor state. Several aggregation-prone variants of ß2m have been reported to date. One of these variants is D76N ß2m, which is a naturally occurring amyloidogenic mutant. To elucidate the molecular mechanisms contributing to the enhanced amyloidogenicity of the mutant, we investigated the equilibrium and kinetic transitions of pressure-induced folding/unfolding equilibria in the wild type and D76N mutant by monitoring intrinsic tryptophan and 1-anilino-8-naphthalene sulfonate fluorescence. An analysis of kinetic data revealed that the different folding/unfolding behaviors of the wild type and D76N mutant were due to differences in the activation energy between the unfolded and the intermediate states as well as stability of the native state, leading to more rapid accumulation of IT state for D76N in the refolding process. In addition, the IT state was found to assume more hydrophobic nature. These changes induced the enhanced amyloidogenicity of the D76N mutant and the distinct pathogenic symptoms of patients. Our results suggest that the stabilization of the native state will be an effective approach for suppressing amyloid fibril formation of this mutant.

Revisiting the Rate-Limiting Step of the ANS-Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity.

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS-protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS-protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.

Modelling Förster resonance energy transfer (FRET) using anisotropy resolved multi-dimensional emission spectroscopy (ARMES).

BACKGROUND: Förster Resonance Energy Transfer (FRET) is widely used to study the structure and dynamics of biomolecular systems and also causes the non-linear fluorescence response observed in multi-fluorophore proteins. Accurate FRET analysis, in terms of measuring changes in donor and acceptor spectra and energy transfer efficiency is therefore critical. METHODS: We demonstrate a novel quantitative FRET analysis using anisotropy resolved multidimensional emission spectroscopy (ARMES) in a Human Serum Albumin (HSA) and 1,8-anilinonaphathalene sulfonate (ANS) model. ARMES combines 4D measurement of polarized excitation emission matrices (pEEM) with multivariate data analysis to spectrally resolve contributing fluorophores. Multivariate analysis (Parallel Factor, PARAFAC and restricted Tucker3) was used to resolve fluorophore contributions and for modelling the quenching of HSA emission and the HSA-ANS interactions. RESULTS: pEEM spectra were modelled using Tucker3 which accommodates non-linearities introduced by FRET and a priori chemical knowledge was used to optimise the solution, thus resolving three components: HSA emission, ANS emission from indirect FRET excitation, and ANS emission from direct excitation. Perpendicular emission measurements were more sensitive to indirectly excited acceptor emission. PARAFAC modelling of HSA, donor emission, separated ANS FRET interacting (Tryptophan) and non-interacting (Tyrosine) components. This enabled a new way of calculating quenching constants using the multi-dimensional emission of individual donor fluorophores. CONCLUSIONS: FRET efficiency could be calculated using the multi-dimensional, resolved emission of the interacting donor fluorophores only which yielded higher ET efficiencies compared to conventional methods. GENERAL SIGNIFICANCE: Shows the potential of multidimensional fluorescence measurements and data analysis for more accurate FRET modelling in proteins.

Fluorescence Enhancement by Calixarene Supramolecular Aggregate.

We herein constructed supramolecular assemblies from guanidinocalixarenes and sulfonatocalixarenes by exploiting multiple salt bridge interactions. They encapsulate six different kinds of fluorescent dyes (both cationic and anionic), leading to a fluorescence enhancement that could not be achieved by either single calixarene. As such, this study advances the research on high-performance fluorophores.

Small molecules as potent biphasic modulators of protein liquid-liquid phase separation.

Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell. However, dysregulated LLPS can also facilitate aberrant phase transitions and lead to protein aggregation and disease. Accordingly, there is great interest in identifying small molecules that modulate LLPS. Here, we demonstrate that 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and similar compounds are potent biphasic modulators of protein LLPS. Depending on context, bis-ANS can both induce LLPS de novo as well as prevent formation of homotypic liquid droplets. Our study also reveals the mechanisms by which bis-ANS and related compounds modulate LLPS and identify key chemical features of small molecules required for this activity. These findings may provide a foundation for the rational design of small molecule modulators of LLPS with therapeutic value.

Protective actions of bioactive flavonoids chrysin and luteolin on the glyoxal induced formation of advanced glycation end products and aggregation of human serum albumin: In vitro and molecular docking analysis.

The post-translational modification of proteins by nonenzymatic glycation (NEG) and the accumulation of AGEs are the two underlying factors associated with the long-term pathogenesis in diabetes. Glyoxal (GO) is a reactive intermediate which has the ability to modify proteins and generate AGEs at a faster rate. Human serum albumin (HSA) being the most abundant serum protein has a higher chance to be modified by NEG. The key objective of the present study is to investigate the potency of chrysin and luteolin as antiglycating and antifibrillating agents in the GO-mediated glycation and fibril formation of HSA. AGEs formation were confirmed from the absorption and fluorescence spectral measurements. Both the flavonoids were able to quench the AGEs fluorescence intensity in vitro indicating the antiglycating nature of the molecules. The formation of fibrils in the GO-modified HSA was confirmed by the Thioflavin T (ThT) fluorescence assay and the flavonoids were found to exihibit the antifibrillation properties in vitro. Docking results suggested that both the flavonoids interact with various amino acid residues of subdomain IIA including glycation prone lysines and arginines via non-covalent forces and further stabilized the structure of HSA, which further explains their mechanisms of action as antiglycating and antifibrillating agents.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

A new modulated crystal structure of the ANS complex of the St John's wort Hyp-1 protein with 36 protein molecules in the asymmetric unit of the supercell.

Superstructure modulation, with violation of the strict short-range periodic order of consecutive crystal unit cells, is well known in small-molecule crystallography but is rarely reported for macromolecular crystals. To date, one modulated macromolecular crystal structure has been successfully determined and refined for a pathogenesis-related class 10 protein from Hypericum perforatum (Hyp-1) crystallized as a complex with 8-anilinonaphthalene-1-sulfonate (ANS) [Sliwiak et al. (2015), Acta Cryst. D71, 829-843]. The commensurate modulation in that case was interpreted in a supercell with sevenfold expansion along c. When crystallized in the additional presence of melatonin, the Hyp-1-ANS complex formed crystals with a different pattern of structure modulation, in which the supercell shows a ninefold expansion of c, manifested in the diffraction pattern by a wave of reflection-intensity modulation with crests at l = 9n and l = 9n ± 4. Despite complicated tetartohedral twinning, the structure has been successfully determined and refined to 2.3 Šresolution using a description in a ninefold-expanded supercell, with 36 independent Hyp-1 chains and 156 ANS ligands populating the three internal (95 ligands) and five interstitial (61 ligands) binding sites. The commensurate superstructures and ligand-binding sites of the two crystal structures are compared, with a discussion of the effect of melatonin on the co-crystallization process.

Tocopheryl Succinate-Induced Structural Changes in DPPC Liposomes: DSC and ANS Fluorescence Studies.

Recent studies show that alpha-tocopheryl succinate (TS) exhibits selective toxicity against cancer cells. In this study, we investigated the effect of TS's presence on the physico-chemical and structural properties of DPPC liposomes using fluorescence parameters (intensity, lifetime, and position of emission maximum) of 1-anilino-8-naphtalene sulphonate (ANS), differential scanning calorimetry (DSC) and zeta potential methods. Increasing the TS presence in the DPPC gel phase produced ANS fluorescence enhancement with a hypsochromic shift of the maximum. The zeta potential measurements show an increase in the negative surface charge and confirmed that this process is connected with the hydrophobic properties of dye, which becomes located deeper into the interphase region with a progressing membrane disorder. Temperature dependence studies showed that an increase in temperature increases the ANS fluorescence and shifts the ANS maximum emission from 464 to 475 nm indicating a shift from hydrophobic to a more aqueous environment. In the liquid crystalline phase, the quenching of ANS fluorescence occurs due to the increased accessibility of water to the ANS located in the glycerol region. The DSC results revealed that increasing the presence of TS led to the formation of multicomponent DSC traces, indicating the formation of intermediate structures during melting. The present results confirmed that TS embedded into the DPPC membrane led to its disruption due to destabilisation of its structure, which confirmed the measured biophysical parameters of the membrane.

Cooperativity Between Orthosteric Inhibitors and Allosteric Inhibitor 8-Anilino-1-Naphthalene Sulfonic Acid (ANS) in Cyclin-Dependent Kinase 2.

While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site, where many kinase inhibitors bind. We have previously discovered that 8-anilino-1-naphthalene sulfonic acid (ANS) binds an allosteric pocket in cyclin-dependent kinase 2 (Cdk2). Here, we detail the positive cooperativity between ANS and orthosteric Cdk2 inhibitors dinaciclib and roscovitine, which increase the affinity of ANS toward Cdk2 5-fold to 10-fold, and the relatively noncooperative effects of ATP. We observe these effects using a fluorescent binding assay and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR), where we noticed a shift from fast exchange to slow exchange upon ANS titration in the presence of roscovitine but not with an ATP mimic. The discovery of cooperative relationships between orthosteric and allosteric kinase inhibitors could further the development of selective kinase inhibitors in general.

Alteration of Protein Binding Affinities by Aqueous Two-Phase Systems Revealed by Pressure Perturbation.

Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifunctional proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo.

Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding.

ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential protein substrates. Thus, these molecular machines must demonstrate greater stability than their substrates in order to ensure continued function in essential quality control networks. We report here a role for ATP in the stabilization of the inner membrane YME1L protease. Qualitative fluorescence data derived from protein unfolding experiments with urea reveal non-standard protein unfolding behavior that is dependent on [ATP]. Using multiple fluorophore systems, stopped-flow fluorescence experiments demonstrate a depletion of the native YME1L ensemble by urea-dependent unfolding and formation of a non-native conformation. Additional stopped-flow fluorescence experiments based on nucleotide binding and unfoldase activities predict that unfolding yields significant loss of active YME1L hexamers from the starting ensemble. Taken together, these data clearly define the stress limits of an important mitochondrial protease.

Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency.

ANS Interacts with the Ca2+-ATPase Nucleotide Binding Site.

The binding of 8-anilino-1-naphthalene sulfonate (ANS) to the nucleotide binding domain (N-domain) of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) was studied. Molecular docking predicted two ANS binding modes (BMI and BMII) in the nucleotide binding site. The molecular interaction was confirmed as the fluorescence intensity of ANS was dramatically increased when in the presence of an engineered recombinant N-domain. Molecular dynamics simulation showed BMI (which occupies the ATP binding site) as the mode that is stable in solution. The above was confirmed by the absence of ANS fluorescence in the presence of a fluorescein isothiocyanate (FITC)-labeled N-domain. Further, the labeling of the N-domain with FITC was hindered by the presence of ANS, i.e., ANS was bound to the ATP binding site. Importantly, ANS displayed a higher affinity than ATP. In addition, ANS binding led to quenching the N-domain intrinsic fluorescence displaying a FRET pattern, which suggested the existence of a Trp-ANS FRET couple. Nonetheless, the chemical modification of the sole Trp residue with N-bromosuccinimide (NBS) discarded the existence of FRET and instead indicated structural rearrangements in the nucleotide binding site during ANS binding. Finally, Ca2+-ATPase kinetics in the presence of ANS showed a partial mixed-type inhibition. The Dixon plot showed the ANS-Ca2+-ATPase complex as catalytically active, hence supporting the existence of a functional dimeric Ca2+-ATPase in sarcoplasmic reticulum vesicles. ANS may be used as a molecular platform for the development of more effective inhibitors of Ca2+-ATPase and appears to be a new fluorescent probe for the nucleotide binding site. Graphical Abstract Molecular docking of ANS to the nucleotide binding site of Ca2+-ATPase. ANS fluorescence increase reveals molecular interaction.

Competitive binding assay for biotin and biotin derivatives, based on avidin and biotin-4-fluorescein.

Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidin was pre-blocked to different extents by the biotinylated analyte and a constant amount of biotin-4-fluorescein (B4F) was added, resulting in strong quenching of the B4F. The assay was robust and the shape of the titration curve immediately revealed whether the data were reliable or perturbed by steric hindrance in case of large biotin derivatives. These advantages justified well the 10× higher sample consumption (~0.6nmol) compared to single point assays. The assay was applied to a representative set of small biotin derivatives and validated by cross-control with the well-established 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) binding assay. In comparison to the 2,6-ANS binding assay, the lower precision (±10%) was compensated by the 100-fold higher sensitivity and the deviations from the ANS assay were ≤5%. In comparison to the more sensitive biotin group assays, the new assay has the advantage of minimal bias for different biotin derivatives. In case of biotinylated DNA with 30 nucleotides, steric hindrance was found to reduce the accuracy of biotin group determination; this problem was overcome by partial digestion to n≤5 nucleotide residues with a 3'-exonuclease. The newly proposed biotin group assay offers a useful compromise in terms of sensitivity, precision, trueness, and robustness.