title: Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis.
abstract: As two important physico-chemical parameters, the acidic and alkaline residues of protein are of evident significance for the evaluation of protein properties and the design of relevant separation and analysis. However, there is still no electrophoretic method used for the direct detection of free acidic and alkaline residues of protein. Herein, we developed the concepts of moving reaction boundary (MRB) and MRB titration, relevant MRB titration theory, and the method of microdevice electrophoresis for the determination of free acidic and alkaline residues of protein. In the MRB titration, the boundary was created with acid or alkali and target protein immobilized via highly cross-linked polyacrylamide gel (PAG). It was theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system. As a proof of concept, seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration. The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database, demonstrating the feasibility of the developed concept, theory and technique. The general methodology of MRB titration has potential application for inexpensive, facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.
authors.affiliation: Laboratory of Bioseparation and Analytical Biochemistry, State Key Laboratory of Microbial Metabolism, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Hou-yu
authors.initials: HY
authors.lastname: Wang
authors.forename: Si
authors.initials: S
authors.lastname: Li
authors.forename: Yun-yun
authors.initials: YY
authors.lastname: Tang
authors.forename: Jing-yu
authors.initials: JY
authors.lastname: Dong
authors.forename: Liu-yin
authors.initials: LY
authors.lastname: Fan
authors.forename: Cheng-xi
authors.initials: CX
authors.lastname: Cao
mesh_terms: D000818:Animals; D002417:Cattle; D004586:Electrophoresis / Q000295:instrumentation*; D004867:Equipment Design; D005240:Feasibility Studies; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055616:Microtechnology / Q000295:instrumentation*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*
chemical_list: D011506:Proteins