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H-Glu(EDANS)-Pro-Leu-Phe-Ala-Glu-Arg-Lys(DABCYL)-OH is an internally quenched substrate for calpain-1 (µ-calpain) with optimal cleavage motifs flanking the scissile bond. The turnover rate of the enzyme to hydrolysis of this FRET substrate based on the PLFAER amino acid sequence is more than 18 times higher than that of EVYGMM derived from the natural substrate α-spectrin cleavage site.