Q223R variant suggested that heterogeneity in association between this mutation and body weight in human populations may be a ributable to differences in study design and power. LepR contains two extracellular CRH domains separated by an immunoglobulin-like domain. Two fibronectin type 3 domains separate the CRH2 domain from a single transmembrane region and a long intracellular signaling region containing a membrane-proximal box 1 motif. The LepR Q223R mutation results from an A to G transversion encoding a glutamine to arginine substitution in the N-terminal CRH1 domain. It has been shown that the CRH2 domain is both necessary and sufficient for leptin binding; while CRH1 is dispensable for a high affinity interaction. To test the hypothesis that the Q223R amino acid replacement affects ligand-binding kinetics of the leptin receptor, we measured the leptin/LepR interaction using SPR technology on a BiaCore T200 platform. To measure leptin association and dissociation for both the Tetrahydroberberine mutant and wild type receptors, the extracellular domain of each receptor was expressed as an Fc-fused chimera and immobilized to a dextran matrix coated with anti-IgG antibody. Increasing concentrations of leptin were then injected into the system. The rates of binding and dissociation were monitored at each leptin concentration in real-time as changes in the refractory index of the capture surface measured in response units. The ability to monitor these interactions in real-time allowed for the determination of binding kinetic constants by global fi ing to a model of 1:1 ligand to receptor binding. The most important finding of this work is that the common Q223R encoding SNP in the LepR extracellular domain does not affect the rates at which leptin binds to and dissociates from its receptor. We investigated whether the Q223R substitution could alter ligand binding using surface plasmon resonance. Because it is both common and non-conservative, the Q223R amino acid replacement has been among the most studied variants of LepR. However, the functional consequences of this variant remain poorly defined. This mutation occurs in the CRH1 domain, which is dispensable for high affinity leptin binding, but may have roles in downstream signaling, receptor trafficking, or surface Eleutheroside-E expression. Stratigopoulos et al. observed no difference in adiposity between WT and Q223R isogenic mice fed a high fat diet. A recent study found no significant association between this LepR polymorphism and obesity in humans. However, the homozygous Q223R encoding genotype did correlate strongly with increased serum cholesterol and low density lipoprotein in both obese and non-obese subjects. In addition, our earlier work has demonstrated a dramatic association between the human Q223R LepR mutation and amebiasis, a common cause of diarrheal morbidity and mortality among children globally. Furthermore, patients carrying the Q223R mutation are at increased risk of Clostridium difficile colitis. SPR was used to measure, in real-time, the interaction between murine leptin and the extracellular domain of murine LepR with or without the Q223R mutation. Until recently, the two primary models of leptin binding have suggested leptin:LepR stoichiometries of 2:2 or 2:4.