Peroxiredoxins as biomarkers of oxidative stress

Background

Peroxiredoxins (Prxs) are a class of abundant thiol peroxidases that degrade hydroperoxides to water. Prxs are sensitive to oxidation, and it is hypothesized that they also act as redox sensors. The accumulation of oxidized Prxs may indicate disruption of cellular redox homeostasis.

Scope of review

This review discusses the biochemical properties of the Prxs that make them suitable as endogenous biomarkers of oxidative stress, and describes the methodology available for measuring Prx oxidation in biological systems.

Major conclusions

Two Prx oxidation products accumulate in cells under increased oxidative stress: an intermolecular disulfide and a hyperoxidized form. Methodologies are available for measuring both of these redox states, and oxidation has been reported in cells and tissues under oxidative stress from external or internal sources.

General significance

Monitoring the oxidation state of Prxs provides insight into disturbances of cellular redox homeostasis, and complements the use of exogenous probes of oxidative stress. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Measuring chlorine bleach in biology and medicine

Background

Chlorine bleach, or hypochlorous acid, is the most reactive two-electron oxidant produced in appreciable amounts in our bodies. Neutrophils are the main source of hypochlorous acid. These champions of the innate immune system use it to fight infection but also direct it against host tissue in inflammatory diseases. Neutrophils contain a rich supply of the enzyme myeloperoxidase. It uses hydrogen peroxide to convert chloride to hypochlorous acid.

Scope of review

We give a critical appraisal of the best methods to measure production of hypochlorous acid by purified peroxidases and isolated neutrophils. Robust ways of detecting it inside neutrophil phagosomes where bacteria are killed are also discussed. Special attention is focused on reaction-based fluorescent probes but their visual charm is tempered by stressing their current limitations. Finally, the strengths and weaknesses of biomarker assays that capture the footprints of chlorine in various pathologies are evaluated.

Major conclusions

Detection of hypochlorous acid by purified peroxidases and isolated neutrophils is best achieved by measuring accumulation of taurine chloramine. Formation of hypochlorous acid inside neutrophil phagosomes can be tracked using mass spectrometric analysis of 3-chlorotyrosine and methionine sulfoxide in bacterial proteins, or detection of chlorinated fluorescein on ingestible particles. Reaction-based fluorescent probes can also be used to monitor hypochlorous acid during phagocytosis. Specific biomarkers of its formation during inflammation include 3-chlorotyrosine, chlorinated products of plasmalogens, and glutathione sulfonamide.

General significance

These methods should bring new insights into how chlorine bleach is produced by peroxidases, reacts within phagosomes to kill bacteria, and contributes to inflammation. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Antioxidative activity of the olive oil constituent hydroxy-1-aryl-isochromans in cells and cell-free systems

Background

Hydroxy-1-aryl-isochromans (HAIC) are newly emerging natural polyphenolic antioxidants, enriched in extravirgin olive oil, whose antioxidative potency was only scarcely characterized using cell-free systems and cells.

Methods

We characterized the activity of HAIC to inactivate reactive oxygen species (ROS) generated by the xanthine/xanthine oxidase system, mitochondria (rat brain) and neural cells. ROS levels were estimated using ROS-sensitive probes, such as Amplex Red, MitoSOX^RED.

Results

HAIC (with 2, 3 or 4 hydroxyl substituents) effectively scavenge ROS released from mitochondria. EC₅₀ values estimated with mitochondria and submitochondrial particles were around 20 μM. Moreover, in PC12 and cultured neural primary cells, HAIC buffered cytosolic ROS. Although HAIC permeate biological membranes, HAIC fail to buffer matrix ROS in isolated mitochondria. We show that hydrogen peroxide was effectively abolished by HAIC, whereas the production of superoxide was not affected.

Conclusion

HAIC exert high antioxidative activity to reduce hydrogen peroxide. The antioxidative activity of HAIC is comparable with that of the stilbene-like, polyphenolic resveratrol, but much higher than that of trolox, N-acetylcysteine or melatonin.

General significance

Unlike resveratrol, HAIC do not impair mitochondrial ATP synthesis or Ca²⁺ retention by mitochondria. Thus, HAIC have the decisive advantage to be potent antioxidants with no detrimental side effects on mitochondrial functions.     

LRRK2, but not pathogenic mutants,protects against H2O2 stress depending on mitochondrial function and endocytosis in a yeast model

Background

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). Studies in the yeast Saccharomyces cerevisiae have provided valuable insights into the mechanisms of cellular dysfunction associated with the expression of faulty PD genes.

Methods

We developed a yeast model for full-length LRRK2 studies. We expressed wild-type (wt) LRRK2 and mutations and evaluated their role during oxidative stress conditions. The involvement of mitochondria was assessed by using rho-zero mutants and by evaluating reactive oxygen species (ROS) production and mitochondrial membrane potential by flow cytometry. The involvement of endocytosis was also studied by testing several endocytic mutants and by following the vacuolar delivery of the probe FM4-64.

Results

Expression of LRRK2 in yeast was associated to increased hydrogen peroxide resistance. This phenotype, which was dependent on mitochondrial function, was not observed for PD-mutants G2019S and R1441C or in the absence of the kinase activity and the WD40 repeat domain. Expression of the pathogenic mutants stimulated ROS production and increased mitochondrial membrane potential. For the PD-mutants, but not for wild-type LRRK2, endocytic defects were also observed. Additionally, several endocytic proteins were required for LRRK2-mediated protection against hydrogen peroxide.

Conclusions

Our results indicate that LRRK2 confers cellular protection during oxidative stress depending on mitochondrial function and endocytosis.

General significance

Both the loss of capacity of LRRK2 pathogenic mutants to protect against oxidative stress and their enhancement of dysfunction may be important for the development of PD during the aging process.     

Genetically encoded fluorescent redox sensors

Background

Life is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes.

Scope of review

In this review, we describe the main types of genetically encoded redox probes, their essential properties, advantages and disadvantages. We also provide an overview of the most important, in our opinion, results obtained using these probes. Finally, we discuss redox-dependent photoconversions of GFP and other prospective directions in redox probe development.

Major conclusions

Fluorescent protein-based redox probes have important advantages such as high specificity, possibility of transgenesis and fine subcellular targeting. For proper selection of a redox sensor for a particular model, it is important to understand that HyPer and roGFP2-Orp1 are the probes for H₂O₂, whereas roGFP1/2, rxYFP and roGFP2-Grx1 are the probes for GSH/GSSG redox state. Possible pH changes should be carefully controlled in experiments with HyPer and rxYFP.

General significance

Genetically encoded redox probes are the only instruments allowing real-time monitoring of reactive oxygen species and thiol redox state in living cells and tissues. We believe that in the near future the palette of FP-based redox probes will be expanded to red and far-red parts of the spectrum and to other important reactive species such as NO, O₂ and superoxide. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Detection of electrophile-sensitive proteins

Background

Redox signaling is an important emerging mechanism of cellular function. Dysfunctional redox signaling is increasingly implicated in numerous pathologies, including atherosclerosis, diabetes, and cancer. The molecular messengers in this type of signaling are reactive species which can mediate the post-translational modification of specific groups of proteins, thereby effecting functional changes in the modified proteins. Electrophilic compounds comprise one class of reactive species which can participate in redox signaling. Electrophiles modulate cell function via formation of covalent adducts with proteins, particularly cysteine residues.

Scope of review

This review will discuss the commonly used methods of detection for electrophile-sensitive proteins, and will highlight the importance of identifying these proteins for studying redox signaling and developing novel therapeutics.

Major conclusions

There are several methods which can be used to detect electrophile-sensitive proteins. These include the use of tagged model electrophiles, as well as derivatization of endogenous electrophile–protein adducts.

General significance

In order to understand the mechanisms by which electrophiles mediate redox signaling, it is necessary to identify electrophile-sensitive proteins and quantitatively assess adduct formation. Strengths and limitations of these methods will be discussed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

A non-toxic fluorogenic dye for mitochondria labeling

Background

Mitochondria, powerhouses of cells, are responsible for many critical cellular functions, such as cell energy metabolism, reactive oxygen species production, and apoptosis regulation. Monitoring mitochondria morphology in live cells temporally and spatially could help with the understanding of the mechanisms of mitochondrial functional regulation and the pathogenesis of mitochondria-related diseases.

Methods

A novel non-cytotoxic fluorogenic compound, AcQCy7, was developed as a mitochondria-specific dye.

Results

AcQCy7 emitted no fluorescent signal outside of cells, but it became fluorescent after intracellular hydrolysis of the acetyl group. The hydrolyzed fluorescent product was well retained in mitochondria, enabling long-lasting fluorescence imaging of mitochondria without cell washing. A 2-day culture study using AcQCy7 showed no sign of cytotoxicity, whereas a commonly used mitochondria-staining probe, Mitochondria Tracker Green, caused significant cell death even at a much lower concentration. Apoptosis-causing mitochondria fission was monitored clearly in real time by AcQCy7.

Conclusions

A simple add-and-read mitochondria specific dye AcQCy7 has been validated in various cell models. Bright mitochondria specific fluorescent signal in treated cells lasted several days without noticeable toxicity.

General Significance

The probe AcQCy7 has been proofed to be a non-toxic agent for long-term mitochondria imaging.     

DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein

Background

Thiol-mediated redox regulation of proteins plays a key role in many cellular processes.

Methods

To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal).

Results

DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions.

Conclusions

DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein.

General significance

The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.     

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells

Background

Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs.

Methods

Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide.

Results

We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs.

Conclusions

Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state.

General significance

These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Using exomarkers to assess mitochondrial reactive species in vivo

Background

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Scope of review

One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.

Major conclusions and general significance

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Gel-based methods in redox proteomics

Background

The key to understanding the full significance of oxidants in health and disease is the development of tools and methods that allow the study of proteins that sense and transduce changes in cellular redox. Oxidant-reactive deprotonated thiols commonly operate as redox sensors in proteins and a variety of methods have been developed that allow us to monitor their oxidative modification.

Scope of the review

This outline review specifically focuses on gel-based methods used to detect, quantify and identify protein thiol oxidative modifications. The techniques we discuss fall into one of two broad categories. Firstly, methods that allow oxidation of thiols in specific proteins or the global cellular pool to be monitored are discussed. These typically utilise thiol-labelling reagents that add a reporter moiety (e.g. affinity tag, fluorophore, chromophore), in which loss of labelling signifies oxidation. Secondly, we outline methods that allow specific thiol oxidation states of proteins (e.g. S-sulfenylation, S-nitrosylation, S-thionylation and interprotein disulfide bond formation) to be investigated.

Major conclusions

A variety of different gel-based methods for identifying thiol proteins that are sensitive to oxidative modifications have been developed. These methods can aid the detection and quantification of thiol redox state, as well as identifying the sensor protein.

General significance

By understanding how cellular redox is sensed and transduced to a functional effect by protein thiol redox sensors, this will help us better appreciate the role of oxidants in health and disease. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Mitochondrial proteolysis: Its emerging roles in stress responses

Background

Mitochondria are multifunctional organelles that not only serve as cellular energy stores but are also actively involved in several cellular stress responses, including apoptosis. In addition, mitochondria themselves are also continuously challenged by stresses such as reactive oxygen species (ROS), an inevitable by-product of oxidative phosphorylation. To exert various functions against these stresses, mitochondria must be equipped with appropriate stress responses that monitor and maintain their quality.

Scope of review

Interestingly, increasing evidence indicates that mitochondrial proteolysis has important roles in mitochondrial and cellular stress responses. In this review, we summarize current advances in mitochondrial proteolysis-mediated stress responses.

Major conclusions

Mitochondrial proteases do not only function as surveillance systems of protein quality control by degrading unfolded proteins but also regulate mitochondrial stress responses by processing specific mitochondrial proteins.

General significance

Studies on the regulation of mitochondrial proteolysis-mediated stress responses will provide the novel mechanistic insights into the stress response research fields.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

Chemical aspects of hydrogen sulfide measurements in physiological samples

Background

Owing to recent discoveries of many hydrogen sulfide-mediated physiological processes, sulfide biology is in the focus of scientific research. However, the promiscuous chemical properties of sulfide pose complications for biological studies, which led to accumulation of controversial observations in the literature.

Scope of review

We intend to provide an overview of fundamental thermodynamic and kinetic features of sulfide redox- and coordination-chemical reactions and protonation equilibria in relation to its biological functions. In light of these chemical properties we review the strengths and limitations of the most commonly used sulfide detection methods and recently developed fluorescent probes. We also give a personal perspective on blood and tissue sulfide measurements based on proposed biomolecule–sulfide interactions and point out important chemical aspects of handling sulfide reagent solutions.

Major conclusions

The diverse chemistries of sulfide detection methods resulted in orders of magnitude differences in measured physiological sulfide levels. Investigations that were aimed to dissect the underlying molecular reasons responsible for these controversies made the important recognition that there are large sulfide reserves in biological systems. These sulfide pools are tightly regulated in a dynamic manner and they are likely to play a major role in regulation of endogenous-sulfide-mediated biological functions and avoiding toxic side effects.

General significance

Working with sulfide is challenging, because it requires considerable amounts of chemical knowledge to adequately handle reagent sulfide solutions and interpret biological observations. Therefore, we propose that a rigorous chemical approach could aid the reconciliation of the increasing number of controversies in sulfide biology. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Genetically encoded fluorescent indicator for imaging NAD/NADH ratio changes in different cellular compartments

Background

The ratio of NAD⁺/NADH is a key indicator that reflects the overall redox state of the cells. Until recently, there were no methods for real time NAD⁺/NADH monitoring in living cells. Genetically encoded fluorescent probes for NAD⁺/NADH are fundamentally new approach for studying the NAD⁺/NADH dynamics.

Methods

We developed a genetically encoded probe for the nicotinamide adenine dinucleotide, NAD(H), redox state changes by inserting circularly permuted YFP into redox sensor T-REX from Thermus aquaticus. We characterized the sensor in vitro using spectrofluorometry and in cultured mammalian cells using confocal fluorescent microscopy.

Results

The sensor, named RexYFP, reports changes in the NAD⁺/NADH ratio in different compartments of living cells. Using RexYFP, we were able to track changes in NAD⁺/NADH in cytoplasm and mitochondrial matrix of cells under a variety of conditions. The affinity of the probe enables comparison of NAD⁺/NADH in compartments with low (cytoplasm) and high (mitochondria) NADH concentration. We developed a method of eliminating pH-driven artifacts by normalizing the signal to the signal of the pH sensor with the same chromophore.

Conclusion

RexYFP is suitable for detecting the NAD(H) redox state in different cellular compartments.

General significance

RexYFP has several advantages over existing NAD⁺/NADH sensors such as smallest size and optimal affinity for different compartments. Our results show that normalizing the signal of the sensor to the pH changes is a good strategy for overcoming pH-induced artifacts in imaging.     

Selective binding interactions of deramciclane to the genetic variants of human α1-acid glycoprotein

Background

α₁-Acid glycoprotein (AGP) plays a decisive role in the serum protein binding of several drugs.Genetic variants of AGP have different ligand binding properties. The binding of deramciclane (DER), a chiral anxiolytic agent, has been studied on A and F1/S genetic variants of AGP.

Methods

The effects of DER and reference drugs on the binding of specific fluorescent and circular dichroism (CD) probes of AGP were determined. Dicumarol (DIC) binding was measured by CD and equilibrium dialysis.

Results

DER effectively displaced probes bound to variant A, while it was less effective at displacing probes bound to variant F1/S. DER increased the binding and inverted the induced CD spectrum of DIC in the solution of variant F1/S. This phenomenon could not be brought about by the enantiomer of DER.

Conclusion

DER has high-affinity binding (K_a ≥ 2×10⁶ M^-1) to variant A, while its binding to the variant F1/S is about thirty times weaker. During simultaneous binding of DER and DIC to variant F1/S a ternary complex having about four times higher affinity is formed, in which the opposite chiral conformation of DIC is favored.

General significance

The binding interactions found prove that AGP can simultaneously accommodate different ligand molecules. Even weakly bound ligands can provoke unexpected allosteric protein binding interactions.