The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.

The measurement of values of apparent equilibrium constants K\' for enzyme-catalyzed reactions involve a substantial number of critical details, neglect of which could lead to systematic errors. Here, interferences, impurities in the substances used, and failure to achieve equilibrium are matters of substantial consequence. Careful reporting of results is of great importance if the results are to have archival value. Thus, attention must be paid to the identification of the substances, specification of the reaction(s), the conditions of reaction, the definition of the equilibrium constant(s) and standard states, the use of standard nomenclature, symbols, and units, and uncertainties. This document contains a general discussion of various aspects of these equilibrium measurements as well as STRENDA (Standards for Reporting Enzymology Data) recommendations regarding the measurements and the reporting of results.

Single molecule fluorescence localization microscopy provides molecular localization with a precision in the tens of nanometer range in the plane perpendicular to the light propagation. This opens the possibility to count molecules and correlate their locations, starting from a map of the actual positions in a single molecule super resolution image. Considering molecular pair correlation as an indication of interaction, and a way to discern them from free molecules, we describe a method to calculate thermodynamic equilibrium constants. In this work, we use as a test system two complementary homo-oligonucleotides, one strand marked with Cyanine 3.5 and the other with Alexa Fluor 647. Hybridization is controlled by the amount of each strand, temperature, and the ionic force, and measured in steady state emission. The same samples are examined in Stochastic Optical Reconstruction Microscopy (STORM) experiments with split-field simultaneous two-colour detection. The effect of multiblinking, labelling-detection efficiency, and determination of the critical distance for association are discussed. We consistently determine values in STORM coincident with those of the bulk experiment.

The oxazole yellow dye, YOYO-1 (a symmetric homodimer), is a commonly used molecule for staining DNA. We applied the brightness analysis to study the intercalation of YOYO-1 into the DNA. We distinguished two binding modes of the dye to dsDNA: mono-intercalation and bis-intercalation. Bis-intercalation consists of two consecutive mono-intercalation steps, characterised by two distinct equilibrium constants (with the average number of base pair per binding site equals 3.5): K1=3.36±0.43×107M-1 and K2=1.90±0.61×105M-1, respectively. Mono-intercalation dominates at high concentrations of YOYO-1. Bis-intercalation occurs at low concentrations.

Selective removal of phenolic compounds (PCs) from de-oiled sunflower kernel is generally considered a key step for food applications, but this often leads to protein loss. PC removal yield and protein loss were assessed during an aqueous or aqueous ethanol washing process with different temperatures, pH-values and ethanol contents. PC yield and protein loss increased when the ethanol content was < 60% or when a higher temperature was applied. Our main finding is that preventing protein loss should be the key objective when selecting process conditions. This can be achieved using solvents with high ethanol content. Simulation of the multi-step exhaustive process showed that process optimization is possible with additional washing steps. PC yield of 95% can be achieved with only 1% protein loss using 9 steps and 80% ethanol content at 25℃. The functional properties of the resulting concentrates were hardly altered with the use of high ethanol solvents.

Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches to attribute the measured quantities to the nature of specific domains of the proteins. Motivated by the common goal in cell signaling research aimed at identifying the domains responsible for particular intermolecular interactions, here we describe a framework for determining the local physicochemical properties of cellular proteins associated with immobile scaffolds. To validate this new approach, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created, and the difference among them in FRAP response is analyzed. We demonstrate that transgelin-2 in actin stress fibers (SFs) interacts with F-actin via two separate domains, and the chemical properties are determined for the individual domains. Its pure diffusion properties independent of the association to F-actin is also obtained. Our approach will thus be useful, as presented here for transgelin-2, in addressing the signaling mechanism of cellular proteins associated with SFs.

Immunoassays are nowadays being employed for rapid contaminant analysis in clinical, environmental, and agrochemical samples. A thorough characterization of the antibody‒antigen interaction can bring light to the immunoreagent selection process in order to develop sensitive and robust tests. Thus, determination of equilibrium and reaction rate constants is usually recommendable. However, this can be quite tricky for low molecular weight compounds, and competitive strategies are commonly followed to estimate apparent affinity values. In the present study, a collection of monoclonal antibodies to penthiopyrad was raised for the first time, and apparent equilibrium constants were assessed by the Langmuir model using three different competitive enzyme-linked immunosorbent assay formats. The obtained KD values from antibody-coated assays were quite close to the corresponding KD values calculated from surface plasmon resonance (SPR) evaluation. These studies were employed to select a pair of immunoreagents for immunoassay development. The KD value for penthiopyrad of the selected antibody obtained by SPR was 0.28 nM. The optimized direct assay showed an IC50 value for penthiopyrad of 0.42 nM (0.15 ng mL-1) in buffer. The limit of quantification for grape, must, and wine samples was 10 ng mL-1. An excellent correlation was found when immunochemical results were compared with those from LC-MS/MS. As an application case, it was determined that 58% of penthiopyrad was still found in wine after fermentation.

Selenium-modified nucleosides are powerful tools to study the structure and function of nucleic acids and their protein interactions. The widespread application of 2-selenopyrimidine nucleosides is currently limited by low yields in established synthetic routes. Herein, we describe the optimization of the synthesis of 2-Se-uridine and 2-Se-thymidine derivatives by thermostable nucleoside phosphorylases in transglycosylation reactions using natural uridine or thymidine as sugar donors. Reactions were performed at 60 or 80 °C and at pH 9 under hypoxic conditions to improve the solubility and stability of the 2-Se-nucleobases in aqueous media. To optimize the conversion, the reaction equilibria in analytical transglycosylation reactions were studied. The equilibrium constants of phosphorolysis of the 2-Se-pyrimidines were between 5 and 10, and therefore differ by an order of magnitude from the equilibrium constants of any other known case. Hence, the thermodynamic properties of the target nucleosides are inherently unfavorable, and this complicates their synthesis significantly. A tenfold excess of sugar donor was needed to achieve 40-48 % conversion to the target nucleoside. Scale-up of the optimized conditions provided four Se-containing nucleosides in 6-40 % isolated yield, which compares favorably to established chemical routes.

Enzymology is concerned with the study of enzyme structure, function, regulation and kinetics. It is an interdisciplinary subject that can be treated as an exclusive sphere of exhaustive inquiry within mathematical, physico-chemical and biological sciences. Hence, teaching of enzymology, in general, and enzyme kinetics, in particular, should be undertaken in an interdisciplinary manner for a holistic appreciation of this subject. Further, analogous examples from everyday life should form an integral component of the teaching for an intuitive grasp of the subject matter. Furthermore, simulation-based appreciation of enzyme kinetics should be preferred over simplifying assumptions and approximations of traditional enzyme kinetics teaching. In this Words of Advice, I outline the domain depth of enzymology across the various disciplines and provide initial ideas on how appropriate analogies can provide firm insights into the subject. Further, I demonstrate how an intuitive feel for the subject can help not only in grasping abstract concepts but also extending it in experimental design and subsequent interpretation. Use of simulations in grasping complex concepts is also advocated given the advantages this medium offers over traditional approaches involving images and molecular models. Furthermore, I discuss the merits of incorporating the historical backdrop of major discoveries in enzymological teaching. We, at AstraZeneca, have experimented with this approach with the desired outcome of generating interest in the subject from people practising diverse disciplines.

Thirteen buffers were investigated for their effect on the binding of adamantanol to β-cyclodextrin and hydroxypropyl-β-cyclodextrin. Stability constants for the β-cyclodextrin complex ranged from 14,800 to 46,000 M-1, and the binding enthalpies were between -23.2 and -10.4 kJ/mole. Compared to water, the stability constant in seven carboxylic acid buffers (citric acid, maleic acid, fumaric acid, succinic acid, malonic acid, malic acid and tartaric acid) was reduced. All seven buffers exhibited a competitive mechanism. Binding constants for the interaction between β-cyclodextrin and buffers ranged from 4 to 44 M-1, and binding enthalpies were in the range -19 to -11 kJ/mole. There was a relation between the chemical structures of the buffers and their ability to bind to cyclodextrin. All seven buffers had a carbon chain consisting of more than three carbons in the backbone. Hydroxyl groups on the carbon chain decreased the binding affinity. 1H and ROESY NMR spectroscopy supported inclusion of the citric acid into the cyclodextrin cavity, although the results for succinic and maleic acids were ambiguous. The results demonstrated that some buffers can interact with cyclodextrin complexes, and careful considerations are necessary when choosing a buffer for cyclodextrin research.