Absolute Quantification Of Active Protein Based On Surface Plasmon Resonance

Active proteins play important roles in the function regulation of human bodies and attract much interest for use in pharmaceuticals and clinical diagnostics.The concentration value of active protein is the most important data needed to understand molecular interactions.However,the lack of primary methods to analyze active proteins means there is currently no metrology standard for active protein measurement.This has become a barrier in pharmaceutical development and clinical diagnostics,and causes problems in protein activity-based measurements,such as the commutability issue in immunoassays.In recent years,calibration-free concentration analysis(CFCA),which is based on surface plasmon resonance(SPR)technology,has been proposed to determine the active concentration of proteins that have specific binding activity with a binding partner without any higher order standards.Based on the concern for human health,this article starts with the two aspects of genetically modified crops and disease diagnosis,and selects two targets for research,one of them is plant-derived G2-EPSPS transgenic protein,another is human myoglobin(hMYO).Measuring the active concentration of G2-EPSPS and hMYO protein by CFCA was proposed in this study.This method involves optimization of the regeneration buffer and preparation of chip surfaces for appropriate reaction conditions by immobilizing ligands on sensor chips(CM5)via amine coupling.The active concentration of protein samples were then determined by injection of protein samples and running buffer over immobilized and reference chip surfaces at two different flow rates(5 and 100 ?L/min).The resulting active concentrations were 2778.6 nM and 62733.9 nM,respectively.The performance of the proposed method was evaluated,that the within-day precisions were 3.26%?4.59%and 2.50%?4.23%respectively,and the between-day precisions were 8.36%and 3.12%,respectively.It show that this method had good repeatability and reproducibility.The total protein concentrations of G2-EPSPS and hMYO were then determined by isotope dilution mass spectrometry(IDMS),and only 17.89%and 43.03%of the proteins were confirmed to be active,showing the active protein was only a small part of the total protein.Using the determined active concentration,the more reliable constants of association ka,dissociation kd,and equilibrium kD were determined to be 2.18?106 M-1 s-1,5.79× 10-3 s-1,2.65×10-9 M and 2.58 × 106 M-1s-1,2.30 × 10-3 s-1,8.88 × 10-10 M respectively.The measurement principle of the proposed method can be clearly described by equations and the measurement result can be expressed in SI units.Therefore,the proposed method shows promise to become a primary method for active protein concentration measurement,which can benefit the development of certified reference materials for active proteins.