Stopped-flow Spectroscopy Research Articles

Elucidating the mechanism of Danicamtiv on force, kinetics, and myosin structure and function.

Myosin modulators are a novel class of small molecules that alter cardiac contractility. Omecamtiv mecarbil, the first identified myosin activator, showed only modest clinical benefits in systolic heart failure patients. Thus, there is an urgency to develop alternative myosin activators. Danicamtiv (Dani) has emerged as a potential candidate; however, a detailed mechanism is not known. Here, we aim to elucidate the mechanism of Dani on contractile function in pig cardiac muscle. Demembranated ventricular tissues show a significant 0.1 pCa unit increase in calcium sensitivity and 10% increase in maximal force after incubation in 1 µM Dani. The most potent effects occur in submaximal calcium concentrations, leading to a flattening of the force-calcium relationship, suggesting decreased cooperativity. Maximal rates of tension redevelopment are decreased by approximately 60% with Dani. Isolated cardiac myofibrils provide details about contractile kinetics. Experiments with 1 µM Dani show a 49% decrease in fast-phase relaxation kinetics. Slow-phase isometric relaxation exhibits 47% slower crossbridge detachment rate and 34% longer thin filament deactivation. Next, we assess ATP utilization in the crossbridge cycle. Filament sliding velocity slows 55% on addition of 0.5 µM Dani, similar to the effect of ADP on velocity. The effects of Dani and ADP are not additive suggesting a similar mode of action. ATP binding is unaltered up to 10 µM Dani using stopped flow spectroscopy. Results of X-ray diffraction studies of porcine myocardium at rest show an increase in equatorial intensity ratio (I1,1/I1,0) in response to 50 µM Dani, reflecting an increased proximity of myosin heads to the thin filament. In conclusion, we hypothesize that Dani primes the thick filament for activation and alters relaxation through inhibited ATP hydrolysis product release. Future studies will test these hypotheses.

Kinetics of Zn-DNA binding structural changes measured through stopped-flow circular dichroism.

Divalent cations are known to cause measurable changes in the circular dichroism signals measuring DNA structure. Comparing transition metal divalent ions and alkaline divalent ions shows different structures as a function of concentration and ion identity. Here, we measured the time-dependent nature of this divalent ion binding and corresponding structural changes in the DNA structure using stopped-flow circular dichroism spectroscopy (CD). We found measurable differences between structural changes measured during zinc and magnesium ion binding to the DNA. Combined with CD spectral studies, these experiments give insight into the dynamics and kinetics underlying these processes.

Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

The divergent pH dependence of substrate turnover in dehaloperoxidases A and B

The pH-dependent peroxidase activity in both dehaloperoxidases A and B was studied by a kinetic assay, stopped flow spectroscopy, resonance Raman spectroscopy, and high-performance liquid chromatography at pH 5.0, 6.0, and 7.0. At pH 7.0, both isozymes follow the peroxidase ping-pong kinetic model derived from the three-step reaction scheme using the steady-state approximation. However, deviation from standard saturation behavior is observed at pH < 6.0 and [TCP] > 0.7 mM, owing to multiple processes: a) self-inhibition of TCP by internal binding; b) oxidation of the product by a pH- and concentration-dependent secondary reaction; and c) formation of an inactive species known as compound RH in the absence of oxidizable substrate. Although DHP-A and DHP-B differ by only 5 amino acids, they show a complete trend reversal in their observed peroxidase kinetics and product yields. Although at pH 7.0 DHP-B had higher TCP oxidation activity than DHP-A as reported previously, as pH was lowered, DHP-A appeared to have a higher peroxidase activity than DHP-B. This is an unprecedented result. However, the fact that there are multiple processes contributing to both kinetics and yield of TCP oxidation complicates interpretation of these data. Deactivation via compound RH and self-inhibition are pH dependent reactions that compete with substrate oxidation. Compound RH formation was observed to be rapid at low pH. A complete set of control experiments were conducted to differentiate the various contributions to the observed enzyme kinetics.

Triethanolamine-modulated interfacial polymerization toward microcrumpled nanofiltration membranes: Performances and mechanisms

Highly permeable thin-film composite (TFC) nanofiltration (NF) membranes featuring high separation efficiencies are of great importance for purifying surface water and ground water. Increasing the filtration area of NF membranes via accurate control of the interfacial polymerization (IP) is proposed to be an efficient way to achieve this goal. In this work, a triethanolamine (TEOA)-modulated IP was utilized to tailor the IP reaction rate and thus regulate the separation performance of NF membranes. The resultant NF membranes were characterized in terms of chemical structures, morphologies, and separation behaviors in detail. The reaction dynamic of the TEOA-doped IP was evaluated by stopped-flow spectroscopy and molecular dynamics. The results showed that the incorporation of TEOA could efficiently regulate the IP reaction rate through hydrogen bonds, increased viscosity, and acid-neutralization effects. As result, a high-performance NF membrane possessing a microcrumpled structure and excellent separation efficiency for ground water was achieved. Hence, the TEOA-modulated IP method is expected to be a maneuverable approach for tailoring excellent NF membranes for ground water treatments.

Vasodilation of Pre-contracted Porcine Retinal Arteries by Carbonic Anhydrase Inhibitors with Enhanced Lipophilicity

Purpose In this study, we investigated the vasodilation properties on pre-contracted retinal arteries of a restricted series of carbonic anhydrase inhibitors (CAIs) of the sulfonamide type with enhanced lipophilicity, to assess if it affects the potency of CAIs as vasodilators. Methods Carbonic anhydrase (CA) inhibition and in vitro kinetics of the compounds designed and synthesized for testing in this study were assessed by extracting human CA isoform proteins (hCA) from human cells expressing the isoforms of interest, and then measure the affinity of the novel compound for the hCAs by stopped-flow CO2 hydrase spectroscopy. Lipophilicity of compounds was measured by obtaining their octanol-water partition coefficient, expressed as calculated logP. Porcine eyes were obtained from a local abattoir, and the wall tension of porcine retinal arteriole segments dissected from the eyes was measured with small wire vessel myography. The effects of the CA compounds on wall tension were assessed by adding them to the myography bath, after pre-contracting the vessel by prostaglandin analog U-46619. Results All compounds induced vasodilation but at different concentrations. Among the tested compounds the most potent vasodilators were found to be the seleno-compound 4 and sulfur-ether compound 8 with EC50 values of 7.13 × 10−5 and 7.93 × 10−5 M, respectively, whereas the remaining ones induced complete vasodilation at EC50 comprised within the sub millimolar range. Conclusions All the data reported in this study (i.e. results from myography, in vitro kinetics and LogPs) confirm the important role played by several CA isoforms in vasodilation, although the precise mechanism of action still remains to be elucidated.

Comparison of the Role of Protein Dynamics in Catalysis by Dihydrofolate Reductase from E. coli and H. sapiens.

Dihydrofolate reductase (DHFR) is a well-studied, clinically relevant enzyme known for being highly dynamic over the course of its catalytic cycle. However, the role dynamic motions play in the explicit hydride transfer from the nicotinamide cofactor to the dihydrofolate substrate remains unclear because reaction initiation and direct spectroscopic examination on the appropriate time scale for such femtosecond to picosecond motions is challenging. Here, we employ pre-steady-state kinetics to observe the hydride transfer as directly as possible in two different species of DHFR: Escherichia coli and Homo sapiens. While the hydride transfer has been well-characterized in DHFR from E. coli, improvements in time resolution now allow for sub-millisecond dead times for stopped-flow spectroscopy, which reveals that the maximum rate is indeed faster than previously recorded. The rate in the human enzyme, previously only estimated, is also able to be directly observed using cutting-edge stopped-flow instrumentation. In addition to the pH dependence of the hydride transfer rates for both enzymes, we examine the primary H/D kinetic isotope effect to reveal a temperature dependence in the human enzyme that is absent from the E. coli counterpart. This dependence, which appears above a temperature of 15 °C is a shared feature among other hydride transfer enzymes and is also consistent with computational work suggesting the presence of a fast promoting-vibration that provides donor-acceptor compression on the time scale of catalysis to facilitate the chemistry step.

Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O2. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle.

Caught in the act: Monitoring O[sbnd]O bond cleavage in Acylperoxoferric cytochrome P450cam to form compound I in real time

While monitoring the reaction of ferric cytochrome P450cam (Cyp101) with substituted peroxybenzoic acids using rapid-scanning, stopped-flow (RSSF) spectroscopy, an intermediate appears en route to formation of the high-valent moiety known as Compound I [Fe(IV)=O/porphyrin radical cation] that is thought to be the key catalytic species for O-atom transfer to substrate. We have previously suggested (Spolitak, T., Dawson, J.H., Ballou, D.P., J. Biol. Chem.2005, 280, 20,300–20,309) that this species is an acylperoxo-ferric heme adduct that subsequently undergoes OO bond cleavage to generate Compound I. Singular value decomposition analysis of the RSSF data for formation of this intermediate shows that the energy of its Soret absorption peak is sensitive to the electron donor properties of the aryl substituents on the peracid. A linear Hammett correlation plot is seen for the energy of the Soret absorption peak vs. the Hammett σ constant. This correlation requires that the aryl substituents remain as part of the ligand bound to the heme iron, providing direct evidence that the adduct is indeed a ferric acylperoxo derivative. Linear Hammett correlation plots are also seen for both the rate of formation of the intermediate as well as for its conversion to Compound I. It is proposed that the electron donating/withdrawing properties of the aryl-bound substituents affect the electrophilic nature for binding substrate, changing the observed rate of formation for the acylperoxo intermediate, as well as the propensity and stability of the substituted benzoic acid to serve as the leaving group during OO bond cleavage yielding Compound I.

Superfast degradation of micropollutants in water by reactive species generated from the reaction between chlorine dioxide and sulfite

Chlorine dioxide (ClO2) is used as an oxidant or disinfectant in (waste)water treatment, whereas sulfite is a prevalent reducing agent to quench the excess ClO2. This study demonstrated that seven micropollutants with structural diversity could be rapidly degraded in the reaction between ClO2 and sulfite under environmentally relevant conditions in synthetic and real drinking water. For example, carbamazepine, which is recalcitrant to standalone ClO2 or sulfite, was degraded by 55%–80% in 10 s in the ClO2/sulfite process at 30-µM ClO2 and 30-µM sulfite concentrations within a pH range of 6.0–11.0. Results from experiments and a kinetic model supported that chlorine monoxide (ClO·) and sulfate radicals (SO4·–) were generated in the ClO2/sulfite process, while hydroxyl radical generation was insignificant. Apart from radicals, dichlorine trioxide (Cl2O3) was generated and largely contributed to micropollutant degradation, supported by experimental results using stopped-flow spectrometry and quantum chemical calculations. The impacts of pH, sulfite dosage, and water matrix components (chloride, bicarbonate, and natural organic matter) on micropollutant abatement in the ClO2/sulfite process were evaluated and discussed. When treating the real potable water, the concentrations of organic (five regulated disinfection byproducts) and inorganic byproducts (chlorite and chlorate) formed in the ClO2/sulfite process were all below the drinking water standards. This study disclosed fundamental knowledge advancements relevant to the reaction mechanisms between ClO2 and sulfite, and highlighed a novel process to abate micropollutants in water and wastewater.

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 μM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Curvature dependence of BAR protein membrane association and dissociation kinetics

BAR (Bin/Amphiphysin/Rvs) domain containing proteins function as lipid bilayer benders and curvature sensors, and they contribute to membrane shaping involved in cell signaling and metabolism. The mechanism for their membrane shape sensing has been investigated by both equilibrium binding and kinetic studies. In prior research, stopped-flow spectroscopy has been used to deduce a positive dependence on membrane curvature for the binding rate constant, kon, of a BAR protein called endophilin. However, the impact of bulk diffusion of endophilin, on the kinetic binding parameters has not been thoroughly considered. Employing similar methods, and using lipid vesicles of multiple sizes, we obtained a linear dependence of kon on vesicle curvature. However, we found that the observed relation can be explained without considering the local curvature sensing ability of endophilin in the membrane association process. In contrast, the diffusion-independent unbinding rate constant (koff) obtained from stopped-flow measurements shows a negative dependence on membrane curvature, which is controlled/mediated by endophilin-membrane interactions. This latter dependency, in addition to protein–protein interactions on the membrane, explains the selective binding of BAR proteins to highly curved membranes in equilibrium binding experiments.

Pre-Steady-State Reactivity of Peptidylglycine Monooxygenase Implicates Ascorbate in Substrate Triggering of the Active Conformer.

Peptidylglycine monooxygenase (PHM) is essential for the posttranslational amidation of neuroendocrine peptides. An important aspect of the PHM mechanism is the complete coupling of oxygen reduction to substrate hydroxylation, which implies no oxygen reactivity of the fully reduced enzyme in the absence of peptidyl substrates. As part of studies aimed at investigating this feature of the PHM mechanism, we explored pre-steady-state kinetics using chemical quench (CQ) and rapid freeze-quench (RFQ) studies of the fully reduced ascorbate-free PHM enzyme. First, we confirmed the absence of Cu(I)-enzyme oxidation by O2 at catalytic rates in the absence of peptidyl substrate. Next, we investigated reactivity in the presence of the substrate dansyl-YVG. Surprisingly, when ascorbate-free di-Cu(I) PHM was shot against oxygenated buffer containing the dansyl-YVG substrate, <15% of the expected product was formed. Substoichiometric reactivity was confirmed by stopped-flow and RFQ EPR spectroscopy. Product generation reached a maximum of 70% by the addition of increasing amounts of the ascorbate cosubstrate in a process that was not the result of multiple turnovers. FTIR spectroscopy of the Cu(I)-CO reaction chemistry was then used to show that increasing ascorbate concentrations correlated with a substrate-induced Cu(I)M-CO species characteristic of an altered conformation. We conclude that ascorbate and peptidyl substrate work together to induce a transition from an inactive to an active conformation and suggest that the latter may represent the "closed" conformation (Cu-Cu of ∼4 Å) recently observed for both PHM and its sister enzyme DBM by crystallography.

Temperature effects on calcium binding to caseins

The kinetics of binding of calcium ions in molar excess to individual caseins and casein ingredients was studied in pH 6.4 aqueous solutions using stopped-flow absorption spectroscopy. An initial second-order reaction, faster for β-casein than for αs-casein due to lower energy of activation (ΔEa1,β = 8.2 kJ∙mol−1; ΔEa1,α = 18.1 kJ∙mol−1, respectively), is followed by a slower first-order reaction with similar energies of activation (ΔEa2,β = 25.3 kJ∙mol−1 and ΔEa2,α = 20.7 kJ∙mol−1) as determined from temperature dependence of rate between 25 °C and 50 °C. Sodium caseinate reacts faster with calcium than both αs-casein and β-casein in the first reaction of the two consecutive reactions, while the rate of the second falls between αs-casein and β-casein. Global spectral analysis showed the UV–visible spectra of the reaction intermediates of the caseins to be more similar to the final products than to the initial casein reactants. Dynamic and static light scattering indicated decreasing particle sizes and increasing particle surface upon calcium-binding most significantly at low temperatures. The calcium binding to casein was found endothermic by isothermal titration calorimetry. Calcium binding seems to be controlled by enthalpy/entropy compensation corresponding to an isoequilibrium temperature of 38 °C in agreement with binding of calcium to o-phosphoserine rather than to aspartate or glutamate side chains of the caseins. Binding capacity and affinity for calcium to αs-casein and sodium caseinate both increased with increasing temperature in agreement with the endothermic nature of the binding. Decreasing enthalpy of binding for each calcium indicating a decrease in heat capacity of the caseins upon calcium-binding. The small difference between binding enthalpy and energy of activation for association of calcium to αs-casein lead to the conclusion that calcium dissociation goes through an early transition state. The rate of calcium dissociation hardly depends on temperature also explaining why calcium binding to caseins is important for calcium bioaccessibility.

Kinetics of fast reactions analyzed with stopped flow spectroscopy

Many physiologically relevant processes occur on the timescales inaccessible by conventional spectrometers. Kinetics of those reactions, occurring in the millisecond to hundreds of seconds range can be studied using stopped-flow technology. These experiments allow to obtain kinetic rate constants and study mechanisms of complex reactions by detecting different types of optical changes (absorbance, fluorescence, etc). Despite basic principle of rapid mixing of reagents together in an observation cell, and stopping the flow that triggers signal acquisition, sample handling and instrumentation play a key role in data quality.

Delineating the role of the k-loop in the KIF1A chemomechanical cycle

The kinesin-3 KIF1A has three prominent biophysical characteristics: fast velocity, superprocessivity, and low resistance to load. Using a combination of single molecule motility assay and stopped-flow spectroscopy, we measured the discrete steps of the chemomechanical cycle. The fast rear-head detachment suggests that the fast velocity originates from a reduced waiting time in the 2-head-bound state, resulting in KIF1A predominantly residing in the vulnerable 1-head-bound state during its stepping cycle and therefore being susceptible to load; while the superprocessivity arises from the relatively high microtubule affinity in the post-hydrolysis state.

Evidence for a Long-Lived, Cu-Coupled and Oxygen-Inert Disulfide Radical Anion in the Assembly of Metallothionein-3 Cu(I)4-Thiolate Cluster.

The human copper-binding protein metallothionein-3 (MT-3) can reduce Cu(II) to Cu(I) and form a polynuclear Cu(I)4-Cys5-6 cluster concomitant with intramolecular disulfide bonds formation, but the cluster is unusually inert toward O2 and redox-cycling. We utilized a combined array of rapid-mixing spectroscopic techniques to identify and characterize the transient radical intermediates formed in the reaction between Zn7MT-3 and Cu(II) to form Cu(I)4Zn(II)4MT-3. Stopped-flow electronic absorption spectroscopy reveals the rapid formation of transient species with absorption centered at 430-450 nm and consistent with the generation of disulfide radical anions (DRAs) upon reduction of Cu(II) by MT-3 cysteine thiolates. These DRAs are oxygen-stable and unusually long-lived, with lifetimes in the seconds regime. Subsequent DRAs reduction by Cu(II) leads to the formation of a redox-inert Cu(I)4-Cys5 cluster with short Cu-Cu distances (<2.8 Å), as revealed by low-temperature (77 K) luminescence spectroscopy. Rapid freeze-quench Raman and electron paramagnetic resonance (EPR) spectroscopy characterization of the intermediates confirmed the DRA nature of the sulfur-centered radicals and their subsequent oxidation to disulfide bonds upon Cu(II) reduction, generating the final Cu(I)4-thiolate cluster. EPR simulation analysis of the radical g- and A-values indicate that the DRAs are directly coupled to Cu(I), potentially explaining the observed DRA stability in the presence of O2. We thus provide evidence that the MT-3 Cu(I)4-Cys5 cluster assembly process involves the controlled formation of novel long-lived, copper-coupled, and oxygen-stable disulfide radical anion transient intermediates.

Rapid kinetics of calcium dissociation from plant calmodulin and calmodulin-like proteins and effect of target peptides

Calcium (Ca2+) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca2+ signals are decoded into defined plant adaptive responses by different Ca2+ sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca2+ decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca2+ dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca2+-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca2+ sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca2+ binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca2+-decoding and Ca2+-controlled target binding mechanisms in plants.

A New Reaction for Improved Calibration of EPR Rapid-Freeze Quench Times: Kinetics of Ethylene Diamine Tetraacetate (EDTA) Transfer from Calcium(II) to Copper(II).

The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im4]2+→ [Cu(II)EDTA]2-, are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time (t Q ), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV-visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu2+ ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu2+ ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)]2- and tetraimidazole Cu(II) ([Cu(Im)4]2+) in X-band EPR spectra. The results are implemented in a simple high-precision 'recipe' for determining t Q . These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.

Impact of linker histone in the formation of ambochlorin-calf thymus DNA complex: Multi-spectroscopic, stopped-flow, and molecular modeling approaches.

Objective(s):This study aimed to evaluate the role of the linker histone (H1) in the binding interaction between ambochlorin (Amb), and calf thymus DNA (ctDNA) as binary and ternary systems. Materials and Methods:The project was accomplished through the means of absorbance, fluorescence, stopped-flow circular dichroism spectroscopy, viscosity, thermal melting, and molecular modeling techniques.Results:Spectroscopic analysis revealed that although Amb was strongly bound to both ctDNA and ctDNA-H1, it showed a greater tendency to ctDNA in the presence of the linker histone. The obtained thermodynamic parameters revealed that both Amb-ctDNA and Amb-ctDNA-H1 interactions were spontaneous, endothermic, and entropy-favored, and hydrophobic interactions played the main role in the formation and stabilization of complexes. Analysis of the stopped-flow circular dichroism results revealed that the binding process of Amb-ctDNA and Amb-ctDNA-H1 required a time of more than 150 milliseconds to complete. Moreover, Amb-ctDNA complex formation was marginally decelerated in the presence of the linker histone. The docking results suggested that the presence of the linker histone may alter the binding sites of Amb from ctDNA minor grooves to major grooves. Conclusion:All quenching processes were governed by a dynamic mechanism. Additionally, Amb did not stabilize or induce considerable conformational changes in ctDNA and ctDNA-H1 complex upon binding. In silico molecular docking results confirmed that Amb was bound to the double-helical ctDNA and ctDNA-H1 via ctDNA grooves. In summary, some binding properties of the interactions between Amb and ctDNA change in the presence of the linker histone.