Determination of lysozyme activity by a fluorescence technique in comparison with the classical turbidity assay.
The aim of this comparable study was to evaluate two different methods for the assay of lysozyme activity. Lysozyme activity was assayed by turbidimetric and fluorescence-based methods with hen egg white lysozyme as standard. The standard activity of each assay was calibrated in units of activity per mg under the experimental conditions (37 degrees C) so that direct comparison between these two assays could be made. The turbidimetric assay was performed using a 0.36 mg/ml Micrococcus lysodeikticus suspension and a microtiter plate reader capable of analyzing enzyme kinetics at 450 nm, and the linearity, in the range of 2.3-23.8 units/ml, presented a correlation coefficient (R2) as high as 0.9967. The fluorescence-based assay was performed with EnzChek kit using a suspension of Micrococcus lysodeikticus labeled with fluorescein and a fluorescence microplate reader. The linearity in the range of 0.47-5.28 units/ml, presented a correlation coefficient (R2) as high as 0.9881. Thus, the turbidimetric assay provides a simple rapid accurate and specific method for the determination of lysozyme activity but with relatively low sensitivity in comparison with the fluorescence-based method which was demonstrated to be a high sensitivity assay, and hence a reliable applicable technique to determine lysozyme activity at low levels e.g. in cell culture systems.
Helal
R
R
Institute of Pharmacy, Free University of Berlin, Germany.
Melzig
M F
MF
D001681:Biological Assay; D002138:Calibration; D004529:Egg White / Q000737:chemistry; D007202:Indicators and Reagents; D008837:Micrococcus / Q000187:drug effects; D009113:Muramidase / Q000032:analysis* / Q000494:pharmacology; D009391:Nephelometry and Turbidimetry; D012015:Reference Standards; D013050:Spectrometry, Fluorescence
D007202:Indicators and Reagents; D009113:Muramidase