Characterization and possible function of an enigmatic reflector in the eye of the shrimp Litopenaeus vannamei.

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 µm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.

A highly reflective biogenic photonic material from core-shell birefringent nanoparticles.

Spectacular natural optical phenomena are produced by highly reflective assemblies of organic crystals. Here we show how the tapetum reflector in a shrimp eye is constructed from arrays of spherical isoxanthopterin nanoparticles and relate the particle properties to their optical function. The nanoparticles are composed of single-crystal isoxanthopterin nanoplates arranged in concentric lamellae around a hollow core. The spherulitic birefringence of the nanoparticles, which originates from the radial alignment of the plates, results in a significant enhancement of the back-scattering. This enables the organism to maximize the reflectivity of the ultrathin tapetum, which functions to increase the eye's sensitivity and preserve visual acuity. The particle size, core/shell ratio and packing are also controlled to optimize the intensity and spectral properties of the tapetum back-scattering. This system offers inspiration for the design of photonic crystals constructed from spherically symmetric birefringent particles for use in ultrathin reflectors and as non-iridescent pigments.

Use of 6-Methylisoxanthopterin, a Fluorescent Guanine Analog, to Probe Fob1-Mediated Dynamics at the Stalling Fork Barrier DNA Sequences.

Fluorescent nucleic acid base mimics serve as excellent site-specific and real-time reporters of the local and global dynamics. In this work, using the fluorescent guanine mimic 6-methylisoxanthopterin (6-MI), we unravel the differential dynamics of replication fork barrier/terminator sequences (RFB1 and RFB3) mediated by fork blocking protein (Fob1). By strategic and site-specific incorporation of this probe, we show that 6-MI is able to capture the changes in global dynamics exhibited by Fob1 and aids in distinguishing between varied architectural forms like double-stranded DNA versus Holliday junctions (HJs). This is important as these barriers are hotspots for recombination. Fluorescence lifetime and anisotropy decay studies further revealed that Fob1 strongly dampens the dynamics in double-stranded RFB1, and the sequence inherently possesses lesser flexibility in comparison to RFB3. We show that 6-MI can probe the differential oligomeric status of Fob1 in response to various architectures, that is, double-stranded versus HJs. This work highlights the unique advantages of 6-MI as a probe when incorporated in nucleic acid frameworks.

Local DNA Base Conformations and Ligand Intercalation in DNA Constructs Containing Optical Probes.

Understanding local conformations of DNA at the level of individual nucleic acid bases and base pairs is important for elucidating molecular processes that depend on DNA sequence. Here, we apply linear absorption and circular dichroism measurements to the study of local DNA conformations, using the guanine base analog 6-methyl isoxanthopterin (6-MI) as a structural probe. We show that the spectroscopic properties of this probe can provide detailed information about the average local base and basepair conformations as a function of the surrounding DNA sequence. Based on these results we apply a simple theoretical model to calculate the circular dichroism spectra of 6-MI-substituted DNA constructs and show that our model can be used to extract information about how the local conformations of the 6-MI probe are influenced by the local base or basepair environment. We also use this probe to examine the pathway for the insertion (intercalation) of a tethered acridine ligand (9-amino-6-chloro methoxyacridine) into duplex DNA. We show that this model intercalator interacts with duplex DNA by a "displacement insertion intercalation" mechanism, whereby the acridine moiety is inserted into the DNA structure and displaces the base located opposite its attachment site. These findings suggest that site-specifically positioned base analog probes can be used to characterize the molecular and structural details of binding ligand effects on local base stacking and unstacking reactions in single- and double-stranded DNA and thus may help to define the molecular mechanisms of DNA-protein interactions that involve the site-specific intercalation of aromatic amino acid side chains into genomic DNA.

Optically functional isoxanthopterin crystals in the mirrored eyes of decapod crustaceans.

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish, and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied by in situ micro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close-to-physiological conditions. We show that the distal mirror consists of three or four layers of plate-like nanocrystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal-structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen-bonded layers of the molecules result in an extremely high calculated refractive index in the H-bonded plane, n = 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye.

Photophysical Characterization of Enhanced 6-Methylisoxanthopterin Fluorescence in Duplex DNA.

The structure and dynamic motions of bases in DNA duplexes and other constructs are important for understanding mechanisms of selectivity and recognition of DNA-binding proteins. The fluorescent guanine analogue, 6-methylisoxanthopterin 6-MI, is well suited to this purpose as it exhibits an unexpected 3- to 4-fold increase in relative quantum yield upon duplex formation when incorporated into the following sequences: ATFAA, AAFTA, or ATFTA (where F represents 6-MI). To better understand some of the factors leading to the 6-MI fluorescence increase upon duplex formation, we characterized the effect of local sequence and structural perturbations on 6-MI photophysics through temperature melts, quantum yield measurements, fluorescence quenching assays, and fluorescence lifetime measurements. By examining 21 sequences we have determined that the duplex-enhanced fluorescence (DEF) depends on the composition of bases adjacent to 6-MI and the presence of adenines at locations n ± 2 from the probe. Investigation of duplex stability and local solvent accessibility measurements support a model in which the DEF arises from a constrained geometry of 6-MI in the duplex, which remains H-bonded to cytosine, stacked with adjacent bases and inaccessible to quenchers. Perturbation of DNA structure through the introduction of an unpaired base 3' to 6-MI or a mismatched basepair increases 6-MI dynamic motion leading to fluorescence quenching and a reduction in quantum yield. Molecular dynamics simulations suggest the enhanced fluorescence results from a greater degree of twist at the X-F step relative to the quenched duplexes examined. These results point to a model where adenine residues located at n ± 2 from 6-MI induce a structural geometry with greater twist in the duplex that hinders local motion reducing dynamic quenching and producing an increase in 6-MI fluorescence.

Sequence-Dependent Conformational Heterogeneity and Proton-Transfer Reactivity of the Fluorescent Guanine Analogue 6-Methyl Isoxanthopterin (6-MI) in DNA.

The local conformations of individual nucleic acid bases in DNA are important components in processes fundamental to gene regulation. Fluorescent nucleic acid base analogues, which can be substituted for natural bases in DNA, can serve as useful spectroscopic probes of average local base conformation and conformational heterogeneity. Here we report excitation-emission peak shift (EES) measurements of the fluorescent guanine (G) analogue 6-methyl isoxanthoptherin (6-MI), both as a ribonucleotide monophosphate (NMP) in solution and as a site-specific substituent for G in various DNA constructs. Changes in the peak positions of the fluorescence spectra as a function of excitation energy indicate that distinct subpopulations of conformational states exist in these samples on time scales longer than the fluorescence lifetime. Our pH-dependent measurements of the 6-MI NMP in solution show that these states can be identified as protonated and deprotonated forms of the 6-MI fluorescent probe. We implement a simple two-state model, which includes four vibrationally coupled electronic levels to estimate the free energy change, the free energy of activation, and the equilibrium constant for the proton transfer reaction. These parameters vary in single-stranded and duplex DNA constructs, and also depend on the sequence context of flanking bases. Our results suggest that proton transfer in 6-MI-substituted DNA constructs is coupled to conformational heterogeneity of the probe base, and can be interpreted to suggest that Watson-Crick base pairing between 6-MI and its complementary cytosine in duplex DNA involves a "low-barrier-hydrogen-bond". These findings may be important in using the 6-MI probe to understand local base conformational fluctuations, which likely play a central role in protein-DNA and ligand-DNA interactions.

Monitoring translocation of multisubunit RNA polymerase along the DNA with fluorescent base analogues.

Here we describe a direct fluorescence method that reports real-time occupancies of the pre- and post-translocated state of multisubunit RNA polymerase. In a stopped-flow setup, this method is capable of resolving a single base-pair translocation motion of RNA polymerase in real time. In a conventional spectrofluorometer, this method can be employed for studies of the time-averaged distribution of RNA polymerase on the DNA template. This method utilizes commercially available base analogue fluorophores integrated into template DNA strand in place of natural bases. We describe two template DNA strand designs where translocation of RNA polymerase from a pre-translocation to a post-translocation state results in disruption of stacking interactions of fluorophore with neighboring bases, with a concomitant large increase in fluorescence intensity.

Quadruplex priming amplification for the detection of mRNA from surrogate patient samples.

Simple and rapid methods for detecting mRNA biomarkers from patient samples are valuable in settings with limited access to laboratory resources. In this report, we describe the development and evaluation of a self-contained assay to extract and quantify mRNA biomarkers from complex samples using a novel nucleic acid-based molecular sensor called quadruplex priming amplification (QPA). QPA is a simple and robust isothermal nucleic acid amplification method that exploits the stability of the G-quadruplex nucleotide structure to drive spontaneous strand melting from a specific DNA template sequence. Quantification of mRNA was enabled by integrating QPA with a magnetic bead-based extraction method using an mRNA-QPA interface reagent. The assay was found to maintain >90% of the maximum signal over a 4 °C range of operational temperatures (64-68 °C). QPA had a dynamic range spanning four orders of magnitude, with a limit of detection of ~20 pM template molecules using a highly controlled heating and optical system and a limit of detection of ~250 pM using a less optimal water bath and plate reader. These results demonstrate that this integrated approach has potential as a simple and effective mRNA biomarker extraction and detection assay for use in limited resource settings.

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3-methyl isoxanthopterin.

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3-methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI-based QPA is a useful assay for DNA amplification and detection.

Vibrational spectroscopic (FT-IR and FT-Raman) studies, natural bond orbital analysis and molecular electrostatic potential surface of Isoxanthopterin.

The FTIR and FT-Raman spectra of Isoxanthopterin have been recorded in the region 4000-450 and 4000-100 cm(-1), respectively. The optimized geometry, frequency and intensity of the vibrational bands of Isoxanthopterin were obtained by the density functional theory (DFT) using 6-311++G(d,p) basis set. The harmonic vibrational frequencies were scaled and compared with experimental values. The observed and the calculated frequencies are found to be in good agreement. The (1)H and (13)C nuclear magnetic resonance chemical shifts of the molecule were also calculated using the gauge independent atomic orbital (GIAO) method. The UV-visible spectrum was also recorded and compared with the theoretical values. The calculated HOMO and LUMO energies show that charge transfer occurs within molecule. The first order hyperpolarizability (ß0), related properties (ß, α0 and Δα) and the Mulliken charges of the molecule were also computed using DFT calculations. Stability of the molecule arising from hyperconjugative interactions, charge delocalization have been analyzed using natural bond orbital (NBO) analysis. The results show that charge in electron density (ED) in the σ* and π* antibonding orbitals and second order delocalization energies (E2) confirms the occurrence of intramolecular charge transfer (ICT) within the molecule. Information about the charge density distribution of the molecule and its chemical reactivity has been obtained by mapping molecular electrostatic potential surface. In addition, the non-linear optical properties were discussed from the dipole moment values and excitation wavelength in the UV-visible region.

Preparation of chitosan-graft-(ß-cyclodextrin) based sol-gel stationary phase for open-tubular capillary electrochromatography.

A novel open-tubular CEC column coated with chitosan-graft-(ß-CD) (CDCS) was prepared using sol-gel technique. In the sol-gel approach, owing to the 3D network of sol-gel and the strong chemical bond between the stationary phase and the surface of capillary columns, good chromatographic characteristics and unique selectivity in separating isomers were shown. The column efficiencies of 55,000∼163,000 plates/m for the isomeric xanthopterin and phenoxy acid herbicides using the sol-gel-derived CDCS columns were achieved. Good stabilities were demonstrated that the RSD values for the retention time of thiourea and isoxanthopterin were 1.3 and 1.4% (run to run, n = 5), 1.6 and 2.0% (day to day, n = 3), 2.9 and 3.1% (column to column, n = 3), respectively. The sol-gel-coated CDCS columns have shown improved separations of isomeric xanthopterin in comparison with CDCS-bonded capillary column.

Isothermal quadruplex priming amplification for DNA-based diagnostics.

We previously developed a method, known as quadruplex priming amplification (QPA), which permits isothermal amplification of DNA. The assay is based on a DNA quadruplex formed by the GGGTGGGTGGGTGGG (G3T) sequence. G3T has three unique properties that are fundamental for QPA; (i) G3T forms a quadruplex with significantly more favorable thermodynamics than the corresponding DNA duplexes; (ii) removal of guanines at the 3'-end inhibits quadruplex formation; and (iii) incorporated fluorescent nucleotides, such as 2-aminopurine (2AP) or 6-methylisoxanthopterin (6MI), which are quenched by neighboring nucleotides, regain maximum emission upon quadruplex formation. New model studies carried out here with primers missing one, two and three guanines reveal that the driving force for QPA comes from the difference in thermal stability between the primer/template and the product complexes. Primers missing one and two guanines are able to self-dissociate from the template upon elongation, whereas QPA is not observed when the primer lacks three 3'-nucleotides. QPA reaches its maximum rate at temperatures slightly higher than the T(m) of the primer/template complex and is more efficient in the presence of only dGTP. QPA-based assays also revealed that Taq is able to incorporate thymidines opposite template 2AP, while no significant incorporation was observed opposite template 6MI.

Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs.

Quadruplexes are involved in the regulation of gene expression and are part of telomeres at the ends of chromosomes. In addition, they are useful in therapeutic and biotechnological applications, including nucleic acid diagnostics. In the presence of K(+) ions, two 15-mer sequences d(GGTTGGTGTGGTTGG) (thrombin binding aptamer) and d(GGGTGGGTGGGTGGG) (G3T) fold into antiparallel and parallel quadruplexes, respectively. In the present study, we measured the fluorescence intensity of one or more 2-aminopurine or 6-methylisoxanthopterin base analogs incorporated at loop-positions of quadruplex forming sequences to develop a detection method for DNA sequences in solution. Before quadruplex formation, the fluorescence is efficiently quenched in all cases. Remarkably, G3T quadruplex formation results in emission of fluorescence equal to that of a free base in all three positions. In the case of thrombin binding aptamer, the emission intensity depends on the location of the fluorescent nucleotides. Circular dichroism studies demonstrate that the modifications do not change the overall secondary structure, whereas thermal unfolding experiments revealed that fluorescent analogs significantly destabilize the quadruplexes. Overall, these studies suggest that quadruplexes containing fluorescent nucleotide analogs are useful tools in the development of novel DNA detection methodologies.

Electronic transition moments of 6-methyl isoxanthopterin--a fluorescent analogue of the nucleic acid base guanine.

Fluorescent nucleic acid base analogues are important spectroscopic tools for understanding local structure and dynamics of DNA and RNA. We studied the orientations and magnitudes of the electric dipole transition moments (EDTMs) of 6-methyl isoxanthopterin (6-MI), a fluorescent analogue of guanine that has been particularly useful in biological studies. Using a combination of absorption spectroscopy, linear dichroism (LD) and quantum chemical calculations, we identified six electronic transitions that occur within the 25,000-50,000 cm(-1) spectral range. Our results indicate that the two experimentally observed lowest-energy transitions, which occur at 29,687 cm(-1) (337 nm) and 34,596 cm(-1) (289 nm), are each polarized within the plane of the 6-MI base. A third in-plane polarized transition is experimentally observed at 47,547 cm(-1) (210 nm). The theoretically predicted orientation of the lowest-energy transition moment agrees well with experiment. Based on these results, we constructed an exciton model to describe the absorption spectra of a 6-MI dinucleotide-substituted double-stranded DNA construct. This model is in good agreement with the experimental data. The orientations and intensities of the low-energy electronic transitions of 6-MI reported here should be useful for studying local conformations of DNA and RNA in biologically important complexes.

Applying 6-methylisoxanthopterin-enhanced fluorescence to examine protein-DNA interactions in the picomolar range.

Incorporation of fluorescent nucleoside analogues into duplex DNA usually leads to a reduction in quantum yield, which significantly limits their potential use and application. We have identified two pentamer DNA sequences containing 6-methylisoxanthopterin (6-MI) (ATFAA and AAFTA, where F is 6-MI) that exhibit significant enhancement of fluorescence upon formation of duplex DNA with quantum yields close to that of monomeric 6-MI. The enhanced fluorescence dramatically increases the utility and sensitivity of the probe and is used to study protein-DNA interactions of nanomolar specificity in this work. The increased sensitivity of 6-MI allows anisotropy binding measurements to be performed at DNA concentrations of 1 nM and fluorescence intensity measurements at 50 pM DNA. The ATFAA sequence was incorporated into DNA constructs to measure the binding affinity of four different protein-DNA interactions that exhibit sequence-specific and non-sequence-specific recognition. In all cases, the K(d) values obtained were consistent with previously reported values measured by other methods. Time-resolved and steady-state fluorescence measurements demonstrate that 6-MI fluorescence is very sensitive to local distortion and reports on different degrees of protein-induced perturbations with single-base resolution, where the largest changes occur at the site of protein binding.

Excited-state electronic properties of 6-methylisoxanthopterin (6-MI): an experimental and theoretical study.

6-Methylisoxanthopterin (6-MI) is a pteridine-based guanine analog that has a red-shifted absorption and high fluorescence quantum yield. Its Watson-Crick base-pairing and base stacking properties are similar to guanine. The fluorescence quantum yield of 6-MI is sensitive to its nearest neighbors and base stacking, making it a very useful real-time probe of DNA structure. The fundamental photophysics underlying this fluorescence quenching by base stacking is not well understood. We have explored the excited-state electronic structure of the 6-MI in frozen 77 K LiCl glasses using Stark spectroscopy. These measurements yielded the direction and degree of charge redistribution for the S(0)→S(1) transition as manifested in the difference dipole moment, Δµ(01), and difference static polarizability, TrΔα. TDDFT (time-dependent density functional theory) was employed to calculate the transition energy, oscillator strength, and the dipole moments of the ground and lowest optically bright excited state of 6-MI (S(0)→S(1)). The direction of Δµ(01) was assigned in the molecular frame based on the Stark data and calculations. These results suggest that the C4═O and C2-NH(2) groups are electron-deficient in the excited state, a very different outcome compared with guanine. This implies that Watson-Crick hydrogen bonding in 6-MI may be modulated by absorption of a photon so as to strengthen base pairing, if only transiently. Solvatochromism was also obtained for the absorption and emission spectra of 6-MI in various solvents and compared with the Stark spectroscopic results using both the Lippert-Mataga and Bakhshiev models.

Characterization of the 6-methyl isoxanthopterin (6-MI) base analog dimer, a spectroscopic probe for monitoring guanine base conformations at specific sites in nucleic acids.

We here characterize local conformations of site-specifically placed pairs of guanine (G) residues in RNA and DNA, using 6-methyl isoxanthopterin (6-MI) as a conformational probe. 6-MI is a base analog of G and spectroscopic signals obtained from pairs of adjacent 6-MI residues reflect base-base interactions that are sensitive to the sequence context, local DNA conformation and solvent environment of the probe bases. CD signals show strong exciton coupling between stacked 6-MI bases in double-stranded (ds) DNA; this coupling is reduced in single-stranded (ss) DNA sequences. Solvent interactions reduce the fluorescence of the dimer probe more efficiently in ssDNA than dsDNA, while self-quenching between 6-MI bases is enhanced in dsDNA. 6-MI dimer probes closely resemble adjacent GG residues, in that these probes have minimal effects on the stability of dsDNA and on interactions with solvent additive betaine. They also serve as effective template bases, although further polymerase-dependent extension of DNA primers past 6-MI template bases is significantly inhibited. These probes are also used to monitor DNA 'breathing' at model replication forks. The 6-MI dimer probe can serve in many contexts as a useful tool to investigate GG conformations at specific sites within the nucleic acid frameworks of functioning macromolecular machines in solution.

Non-conventional hydrogen bonds: pterins-metal anions.

In this paper, we present an analysis of the interaction of metal ions (Cu, Ag and Au) with three different pterins (pterin, isoxanthopterin and sepiapterin) to provide insights concerning the formation of conventional and non-conventional H bonds. Density functional theory calculations were performed in order to reveal the optimized structures of pterin molecules, dimers and tetramers compounds, both with and without metal anions (M). The interaction with small metal clusters (M(3)) is also considered. The formation of different systems is characterized in terms of the structural parameters and hydrogen binding energies (HBE). The HBE values for pterin-M systems presented in this study lie between 22 and 60 kcal mol(-1) and can therefore be classified as strong conventional and strong non-conventional hydrogen bonds. The HBE with small metal clusters (pterin-M(3)) are smaller than the HBE with metal atoms. Vertical electron detachment energies (VEDEs) are also reported in order to analyze the influence of the hydrogen bond on electronic properties. A direct correlation between VEDEs and HBE was found for pterin-M and pterin-M(3) complexes; i.e. as the VEDEs increase, the HBE also augment. The only exception is with Ag(3). The main conclusion derived from this study is that the strong non-conventional hydrogen bonds formed between pterins, dimers and tetramers do not affect the formation of conventional hydrogen bonds between pterins but they do influence the VEDEs.

Regulation of xanthine oxidase activity by substrates at active sites via cooperative interactions between catalytic subunits: implication to drug pharmacokinetics.

Three xanthine oxidase substrates (i.e., xanthine, adenine, and 2-amino-4-hydroxypterin) show a "substrate inhibition" pattern (i.e., slower turnover rates at higher substrate concentrations), whereas another two substrates (i.e., xanthopterin and lumazine) show a "substrate activation" pattern (i.e., higher turnover rates at higher substrate concentrations). Binding of a 6-formylpterin at one of the two xanthine oxidase active sites slows down the turnover rate of xanthine at the adjacent active site from 17.0 s(-1) to 10.5 s(-1), and converts the V-[S] plot from "substrate inhibition" pattern to a classical Michaelis-Menten hyperbolic saturation pattern. In contrast, binding of xanthine at an active site accelerates the turnover rate of 6-formylpterin at the neighboring active site. The experimental results demonstrate that a substrate can regulate the activity of xanthine oxidase via binding at the active sites; or a xanthine oxidase catalytic subunit can simultaneously serve as a regulatory unit. Theoretical simulation based on the velocity equation derived from the extended Michaelis-Menten model shows that the substrate inhibition and the substrate activation behavior in the V-[S] plots could be obtained by introducing cooperative interactions between two catalytic subunits in homodimeric enzymes. The current work confirms that there exist very strong cooperative interactions between the two catalytic subunits of xanthine oxidase.