Competitive Adsorption Of Multi-component Proteins And Co-immobilization Of Binary-enzymes On CMK-3

Mesoporous carbon materials have been widely employed as carriers for proteins,enzymes and drugs owing to its special surface structure. It has been confirmed thatsequential injection system was a good performance for online preparation of biometricfeatures materials and real-time characterization. In the present work, CMK-3was selected asthe carriers, while bovine serum albumin (BSA), hemoglobin (Hb), immunoglobulin (IgG),urate oxidase (UOD), horseradish peroxidase (HRP) were chosen as the objects for the studyof interaction between proteins (enzymes) and CMK-3in the sequential injectionanalysis–UV/Vis system which has been established. The contents and results are as follows:Part I On-line adsorption and competitive adsorption of proteins on CMK-3The on-line adsorptions of BSA, Hb and IgG on CMK-3were investigated by employedthree operation modes, i.e., the single component adsorption, the sequential adsorption of thetwo-component, simultaneous adsorption of the two-component. The results showed that: thesingle-component adsorption of three proteins on CMK-3was in accordance with theLangmuir adsorption model; The loaded amount of three proteins was IgG> Hb> BSA;Comparing with single-component adsorption, a first-come-first-adsorbed basis was observedby employing the sequential adsorption; It was also confirmed that the competitive adsorptionoccurred in the simultaneous adsorption, the presence of Hb promoted the adsorption of BSA,on the contrary, the presence of Hb inhibited the adsorption of IgG possessing large molecularweight.Part II SIA-Enzymatic assay for the determination of uric acidSequential injection UV/Vis spectrometric single-enzyme/enzyme-coupled method forthe determination of uric acid were established. The results showed that the single-enzymemethod and enzyme-coupled method not only provided a linear range of0.025mmol/L~0.60mmol/L and0.05mmol/L~0.40mmol/L respectively with detection limit of0.01mmol/L,which met the needs of the clinical analysis of uric acid, but also minimized sample andreagent consumption. Compared with the single enzymatic, enzyme coupled method has littleinterference matrix.Part Ⅲ Co-immobilization of binary-enzymes on CMK-3The online immobilization of HRP and UOD on CMK-3using single enzymeimmobilization, the sequential immobilization and co-immobilization were explored. The results showed that: There was high amount immobilization of UOD as well as UOD andHRP on CMK-3; The loading amount of sequential immobilization was equivalent to theprotein sequential adsorption; For immobilized UOD, however, there was almost no catalyticactivity to uric acid. The possible reasons were deduced as the following two: the allantoingenerated by the catalytic reaction between UOD and UA was wrapped in the surface of UODto cover the active sites; the conformation of UOD immobilized on CMK-3was changed.This result implied that CMK-3was not the suitable carrier for urate oxidase.