A New Lysozyme Assay Based on Fluorescence Polarization or Fluorescence Intensity Utilizing a Fluorescent Peptidoglycan Substrate1

A new sensitive, rapid and simple method for lysozyme assay is described which is based on either fluorescence polarization or fluorescence intensity using fluorescein-labeled peptido-glycan as a substrate. The peptidoglycan was obtained from Micrococcus lysodeikticus after extensive digestion with Pronase and washing with Triton X-100 followed by various solvents. Subsequently, it was labeled with fluorescein isothiocyanate (FITC) at the amino group of the peptide. When the FITC-labeled substrate was subjected to lysozy.me digestion, an increase of fluorescence intensity or a decrease of fluorescence polarization value (P value) was ap-parent in five minutes at a lysozyme concentration as low as 0.1 or 0.01 ^g/ml, respectively. The effect of other hydrolytic enzymes including a-mannosidase, proteases and RNase on the P value was found to be negligible. The measured values represented the specificity and dose of lysozyme added. Apparent Vmax and Km values for two different lysozymes, chicken egg white and human, could be determined by this method. Fluorescence polarization has a wide range of applications with its high sensitivity and simplicity, if an adequate instrument is available. For in-stance, it has been applied for many proteins and peptides {2-4). I have also demonstrated, (i) the antigen-antibody reaction of antitumor protein antibiotic neocarzinostatin or of angiotensin I an