Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.
The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteinase inhibitor p-chloromercuribenzoate. The fluorescence emission appears at its maximum steady-state yield immediately on addition of DnsArgH to the HSDM1C1 fibrosarcoma cells. The immediacy of the DnsArgH reaction supports the contention that DnsArgH binding may be to cell surface-associated proteinases. Quantification of the cell proteinase concentration, by comparison of the fluorescence yield obtained from DnsArgH interactions with bovine trypsin and papain, indicates 10(-15) to 10(-16) mol of proteinase per HSDM1C1 cell. In fluorescence microscopy, DnsArgH fluorescence appears distributed throughout the fibrosarcoma cell without association to organelles. DnsArgH fluorescence from normal fibroblast controls (IMR-90) was found to be substantially lower than in the transformed fibrosarcoma cells, supporting a hypothesis that proteinases have a role in malignancy.
D001120:Arginine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D003619:Dansyl Compounds / Q000138:chemical synthesis*; D010450:Endopeptidases / Q000378:metabolism*; D005354:Fibrosarcoma / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006651:Histocytochemistry; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009447:Neuroblastoma / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; C040775:dansylargininal; D001120:Arginine; D010450:Endopeptidases