New fluorescent method for the histochemical detection of tripeptidyl peptidase I using glycyl-l-prolyl-l-met-2-anthraquinonyl hydrazide as substrate.

A new substrate for tripeptidyl peptidase I (TPP I; E.C.3.4.14.9)-Gly-L-Pro-L-Met-2-anthraquinonyl hydrazide (Gly-Pro-Met-2-AH) is synthesized and used for the fluorescent histochemical detection of the enzyme. The enzyme liberates low soluble 2-anthraquinonylhydrazine, which-couples quickly with 3-nitrobenzaldehyde (3-NBA) yielding a highly fluorescent water-insoluble hydrazone--3-nitrobenzylidene-2-anthraquinonylhydrazone. The latter compound is localized precisely at sites of enzymatic activity and marks them with a very bright and stable orange-red fluorescence after excitation with conventional monochromatic andlaser green light (lambda(exc)=520-580 nm). The new technique is used successfully for the visualization of the enzyme in tissue sections of different rat organs - and represent the first fluorescent histochemical method for that peptidase.