Novel Method for Determination of Lysozyme Based on Fluorescence Recovery of a Cationic Aluminum Phthalocyanine-Mucopolysaccharides Association Complex Used as a Red Emitting Fluorogenic Substrate / 分析化学

ABSTRACT

We developed a novel method for the rapid determination of lysozyme using a new fluorogenic substrate that consists of a cationic aluminum phthalocyanine ( tetra ( trimethylammonio ) aluminum phthalocyanine, TTMAAlPc ) , and an anionic mucopolysaccharide ( heparin, HP ) . We found that fluorescence from the cationic aluminum phthalocyanine, a red-region fluorescence probe, was quenched significantly in acidic media in the presence of low concentrations of anionic mucopolysaccharide heparin ( HP) bearing anionic sulfonic acid groups, because of induced aggregation. The practically non-fluorescent substrate degraded into small molecular fragments upon the hydrolysis of lysozyme, and thus the phthalocyanine molecules aggregated in HP were released, resulting in significant fluorescence recovery in the reaction system. This phenomenon forms the foundation of the proposed method. The reaction mechanism was determined using fluorescence spectroscopy and fluorescence anisotropy techniques. Factors that affected the determination were investigated. Under optimal conditions, the linear range was 0. 2-2 mg/L, and the detection limit was 0. 015 mg/L. The developed method is easy to operate and has good selectivity and sensitivity. This method was used in the analysis of practical samples of lysozyme, and the results were in agreement with those determined by a conventional turbidimetric method.