Methods for Measuring Ligand-Receptor Affinity

Introduction 

The life of any organism; be it an ordinary bacterium or multicellular animal greatly depends on various interactions happening between molecules. For instance, selective pairing of various molecules requires genetic materials to replicate accordingly in order for this to be successful. The constant selective interaction between various protein molecules necessitates a well-established cellular design. It is apparent that each and every step involving movement within a particular cell is instigated by macromolecular interactions. In the course of this work specific terms such as ligand will be used to stand for species that have the ability to selectively bind in a stoichiometric and reversible way in reference to a big molecule composition. The molecules in this case can either be in the form of genetic material or protein. On the other hand, it will be shown that the subcellular distributions determine the essence of various biomolecules. Also in the event that someone has two molecules which are not identical though they have some close association, it is possible to label one as a Ligand and the other a Receptor . The latter in this case will be a protein molecule whereas the former will turn out to be a small molecule. In the course of this analysis various methods used in the measurement of Ligand-Receptor-Affinity will be established. 

Equation 

A general equation involving the interaction between various molecules can be indicated as shown below 

D+R= K+1 DR ( Florin, Moy & Gaub, 2014). 

Whereby, D stands for the concentration of the equilibrium that is free and R stands for the universal gas constant. Alternatively the k+1 apply in defining the overall rate of the constants. 

At equilibrium it is implied that the various reactions happen at an equivalent pace. 

Labeled Ligand Binding 

Fluorescent ligand Binding Assays 

The spectrum of wavelengths observable in a fluorescent acts as an enabler in detection of multiple colors and images. While measuring ligand receptor affinity, methods that can be used in this case involve: Anisotropy, polarization and bioluminescence resonance ( Luty et al., 2007 ). The first method can apply where varying intensities of light emanating from a fluorophore are being measured. For instance, a recent study pertaining to protein receptors showed that this is one of the most effective binding assays available ( Luty et al., 2007 ). When establishing the resonance, various labels are placed on amino acids. In the process, the distance between the two labels is effectively analyzed. In this case conformational reformation of proteins interactions can be vividly be depicted ( Luty et al., 2007 ). Further still, in the study of molecular interactions, some of the studies being used currently incorporate multi-channel fluorescence. 

Radio ligand binding 

This method is very useful in the detection of binding to a particular target. The membrane bound molecules are the greatest beneficiaries of this technique. The fundamentality of this initiative emerges where it becomes possible to determine the anatomical distribution of various receptors ( Luty et al., 2007 ). Some of the constituents of the method are: competitive and saturation assays ( Ornitz et al., 2012) . The latter increases the receptor level concentrations in a bid to determine equilibrium binding in the radioactive ligand whereas; the former is responsible for measuring the affinity of tissues as well as determining the equilibrium resulting from binding of radio ligand. 

Bioluminescent binding 

The greatest enablers of these incentives involve high sensitivity and reproducibility, which are extensively applied to BRET analysis. In this case various protein hormones are effectively bound along with their receptors ( Jecklin et al., 2009 ). Alternatively, the movement of G-protein receptors occurs effectively by using this method. In the development of bioluminescent binding assays, various hormone specifics are well conjugated with a NanoLuc reporter ( Jecklin et al., 2009 ). The various proteins are then isolated via chromatography before being analyzed via the bioluminescent technique. A genetic approach to this technique can also be embraced whereby fusion of a bioluminescent reporter occurs ( Jecklin et al., 2009 ). 

Label Free Ligand Binding 

Surface Plasmon Resonance (SPR) 

In a situation whereby electromagnetic waves are produced following the interaction of light with surface charges, a surface Plasmon resonance is initiated. In this case, SPR happens to be one of the most effective techniques of establishing the relationship involving various molecules in reference to kinetics. The process enables the analyzer to determine the interaction existent between a drug and the target ( Peralta, 2006 ). In this case, a ligand-receptor and its dissociation are initiated, closely followed by the exclusion of the ligand. The SPR process establishes a sensorgram which ends up detecting how the various wavelengths vary ( Peralta, 2006 ). Its sensitivity, however, appears to be lower in the detection of conformational change pertaining to binding. It is, nonetheless, reputed for lipid-protein interactions. 

Nano fluidic Fluorescence microscopy 

The technique involves the application of a combination of nanofluidic-based biosensors ( Peralta, 2006 ). These components are used effectively in the kinetic assessment of interactions existing between various proteins. The process makes it possible for binding kinetics to be measured. In addition to this, the process is effective in the analysis of antigen-antibody via a fluorescence microscope. 

Plasmon waveguide resonance 

This method is similar to SPR owing to the utilization of surface excited Plasmon. This technique uses a polarized wave laser to stimulate the electromagnetic waves ( Böhm & Schneider, 2013 ). In addition, the assay immobilizes receptors on the SiO 2 surface ( Böhm & Schneider, 2013 ). Alternatively, conformational changes and mass density are effectively identified. The method is hence very effective in the examination of receptor changes. In addition to this, the assay demonstrates how distant ligands lead to conformational alterations in the receptors. The establishment of biological membranes is one of the major strengths of this initiative in addition to its ability to study ligand binding. 

Whispering gallery micro resonator 

In this case, a variety of micro resonators which exhibit great resonance towards light are applied via this technique. The molecules bound to the surface end up inducing change in regards to the changes in wavelengths. The initiative is advantageous owing to the fact that it offers consistent solution based on bio sensing ( Jecklin et al., 2009 ). In ligand binding, the latter manages to establish equilibrium in various interactions such as those that involve antibodies ( Böhm & Schneider, 2013 ). It has recently been established that the micro resonators are bound to soon become biodegradable. 

Resonant waveguide grating 

The component is used in the determination of various affinities in regards to target analytics via biological immobilization of receptors. Surface linked electromagnetic wave is applied to provide an analysis of interactions occurring in reference to the receptor and ligand ( Purich & Allison, 2010 ). In the process maximum incoupling efficiency ends up being obtained via a resonance angle ( Jacobs & Cuatrecasas, 2011 ). 

Bio layer interferometry biosensor 

The technique is almost similar to the aforementioned one though in this case, a spectrometer is used to detect the patterns of interference. In the process a specific pattern of interference is generated. It is, however, altered in the event when ligands and receptors are bound together. A spectrometer can be used in this case to detect the various alterations occurring. It is a method of binding kinetics in addition to biolayer interferometry ( Maehashi et al., 2007 ). The method is hence one of the most effective ligand binding techniques available. 

Structure Based Binding 

Nuclear magnetic resonance (NMR) 

In the study of protein-ligand complexities no method stands out best compared to the NMR. The technique embraces both protein and ligand based analysis mechanisms. It thus becomes possible to identify the binding sites for ligands through protein resonance and changes in chemical shifts. Alternatively, NMR techniques which depend almost on the entire molecular magnetization shift are observable via a variety of spectroscopy experiments. Methods such as the NOESY technique have the ability of analyzing molecules with low weight levels in a substoichiometric protein quantity ( KENT, 2010 ). Negative NOEs are observable in a sample containing molecules mixed by large amounts of protein which are binding. Non-binding proteins on the other hand, are illustrated by negative NOEs. In addition, the subsequent binding of two ligands on the surface of the protein results in the formation of a complex ( Luty et al., 2007 ). Owing to contemporary advancements in the field of science, there is a possibility for formation of binding sites owing to protein-ligand interactions. 

X-ray crystallography 

A receptor ligand composite is obtained via this technique. This is especially useful for the target-based design. The X-ray crystallography is one of the most commonly used techniques. It relies heavily the ability of the protein molecules to grow. In the process of precipitation, the molecules of proteins are agglutinated forming an ordered sequence in form of a lattice ( Lucas & Larive, 2014 ). In this state, the crystallography initiates diffraction, or what is commonly known as X-ray scattering ( Tembre & Mc Cammon, 2004 ). The crystallographer then manipulates three- dimensional structure of the protein using computer aided designs. 

Thermodynamic Binding 

Thermal denaturation 

This method works via differential scanning fluorimetry (DSF) which involves the denaturing of protein molecules ( Baselt et al., 2008 ). The changes occurring in the various proteins under analysis can be detected through the use of fluorophores. Receptors in this case, interact with ligands and then undergo a structural change which has the ability of accumulating thermal stability ( Lieto, Cush & Thompson, 2013 ). The changes occurring in the entire process are analyzed by thermal denaturation which is effective in chemical profiling of proteins. In addition to this, meltdown a program which involves the examination of a thermal melt curve also plays a critical role in the process. Some of the other viable uses of the technique involve: outlier rejection and performing control checks ( Lieto, Cush & Thompson, 2013 ). 

Isothermal Titration Calorimetry (ITC) 

When a binding reaction occurs, heat is produced and at that point the ITC becomes a useful component of evaluating enthalpy variation resulting from the heat generated. Alternatively, the method is also applied in comparing ligands that exhibit identical binding affinities through the detection of differences that are existent in the various modes of binding. On the other hand, this happens to be the best technique that can enable one to gain the best understanding of thermodynamics pertaining to the binding process ( Florin, Moy & Gaub, 2014 ). Detection occurs in the range of 1nM-100µM. currently, there has been an introduction of an ITC assay that that the ability to initiate characterization of two ligands exhibiting different affinities at once ( Florin, Moy & Gaub, 2014) . 

Whole Cell Ligand Binding 

Surface acoustic wave biosensor (SAW) 

In this type of assay, the amplitudes produced by the acoustic waves are effectively detected. The technique involves the application of a sensor whereby the surface generated from mono-crystalline material becomes excited in order for it to oscillate the waves ( Peralta, 2006 ). Upon the alteration of the mass of various molecules, the phase of the acoustic waves ends up being shifted. When conformational changes of the molecules of proteins occur, a change in the amplitude of the acoustic waves is also introduced ( Peralta, 2006 ). A SAW device is attached capacitively by accommodating electrons which initiate a decrease in the volume of cells flowing through a particular surface. Saw devices are composed of a thin parylene layer whose aim is to create a surface that is chemically homogenous ( Peralta, 2006 ). Further still, owing to its efficiency of use, the technique is applicable in the designation of any form of array. 

RWG biosensor 

In the event of bimolecular interaction analysis, the RWG is one of the best techniques to use in analyzing the interaction. On the other hand, the transformation drug receptor in the cells amounting to phenotypic signatures is a result of the biosensor. In addition to this, it is also reputed for detection of cellular responses that are generated by the cellular events which have the ability to alter the mass density of the sensor ( Peralta, 2006 ). On the other hand, RWG detects perfusion flow that occurs in real time by quantifying the effect of ligand binding ( Lutz & Kenakin, 2009 ). It is also possible to detect the duration taken for the successful recovery of the receptor as well as performing an analysis of the impact created by the protease receptor. 

Conclusion 

The in the measurement of Ligand-Receptor-Affinity is essentially a mechanism which is supported by many methods. As indicated above, the main categories involve: Labeled ligand binding, structured based binding, Thermodynamic binding and Whole Cell Ligand binding. These techniques have emerged as some of the most effective means of measuring Ligand receptor affinity. The first technique can be established through Fluorescent ligand Binding Assays which enable the detection of multiple color images, Radio ligand binding used to determine anatomical distribution of various receptors and bioluminescent binding used to enable the movement of G-protein receptors. In addition, the second technique can be achieved by Surface Plasmon resonance which establishes relationships between various molecules and kinetics, Nano fluidic Fluorescence microscopy which ensures that binding kinetics initiated is successful and Plasmon waveguide resonance which is similar to SPR. Others such as the Nuclear magnetic resonance (NMR) under the structural based model which make it possible to identify binding sites for ligands via protein receptors. There is hence no doubt that the aforementioned techniques make it possible for the relationship between Ligands and Receptors to be established so that it can be possible to measure Ligand-Receptor Affinity. 

References  

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