Metacaspases are cysteine-dependent proteases found in fungi, plants, and protozoa. They are involved in programmed cell death, pathogenicity, and stress responses. Our lab studies the Type Ia metacaspase from the fungus Schizophyllum commune (ScMCA-Ia). These metacaspases require calcium for activation and cleave proteins and peptides after lysine and arginine (Arg) residues in the presence of calcium ions. One method for measuring the degradation activity of metacaspases is to fuse the fluorophore 7-amino-4-methylcoumarin (AMC) to a small peptide, Gly-Gly-Arg in this case. AMC emits fluorescence when the peptide is cleaved between Arg and AMC. As more AMC is released into the reaction mixture, the measured fluorescence increases. This reaction takes place inside a plate reader where the fluorescence can be recorded with excitation at 350 nm and emission at 460 nm. Fluorescence intensity is proportional to the molar concentration of AMC. This project presents a general method for the calibration of fluorescence readings on microplate readers that can be used to convert arbitrary fluorescence units per second, FLU/s, into units of micromolar per second, µM/s. Using known concentrations of protein, we performed kinetic assays to get a range of protein activity in units of fluorescence/second. Upon a series of dilutions of AMC, we obtained AMC concentrations that had the same range of fluorescence values as the activity of our protein. After plotting fluorescence vs. [AMC], we fit the data with a linear regression. The data was analyzed to create a standard curve that can be used for conversion between units. Future researchers can use this to express protein activity in absolute units of molarity/second.
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