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. 1983 Feb 1;209(2):299–307. doi: 10.1042/bj2090299

Rhodamine-based compounds as fluorogenic substrates for serine proteinases.

S P Leytus, L L Melhado, W F Mangel
PMCID: PMC1154094  PMID: 6342611

Abstract

A new fluorogenic substrate for serine proteinases, bis(N-benzyloxycarbonyl-L-argininamido)Rhodamine [(Cbz-Arg-NH)2-Rhodamine], was synthesized, purified and chemically and enzymically characterized. This compound, which employs Rhodamine as a fluorophoric leaving group, is the first in a series of substrates designed to measure the amidase activity of proteinases. Cleavage of one of the amide bonds of (Cbz-Arg-NH)2-Rhodamine by a trypsin-like serine proteinase converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Significant differences in the electronic absorption and fluorescence emission spectra and quantum yields of bis-, mono- and un-substituted Rhodamine are reported. Macroscopic kinetic constants for the interaction of (Cbz-Arg-NH)2-Rhodamine with bovine trypsin, human and dog plasmin and human thrombin were determined. Compared with the corresponding 7-amino-4-methylcoumarin-based analogue, (Cbz-Arg-NH)2-Rhodamine exhibits an increase in sensitivity with these enzymes of 50--300-fold. The physical basis for this increase in sensitivity is discussed.

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Selected References

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  1. Bigbee W. L., Weintraub H. B., Jensen R. H. Sensitive fluorescence assays for urokinase using synthetic peptide 4-methoxy-beta-naphthylamide substrates. Anal Biochem. 1978 Jul 15;88(1):114–122. doi: 10.1016/0003-2697(78)90403-7. [DOI] [PubMed] [Google Scholar]
  2. Castellino F. J., Sodetz J. M. Rabbit plasminogen and plasmin isozymes. Methods Enzymol. 1976;45:273–286. doi: 10.1016/s0076-6879(76)45026-7. [DOI] [PubMed] [Google Scholar]
  3. Castillo M. J., Nakajima K., Zimmerman M., Powers J. C. Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases. Anal Biochem. 1979 Oct 15;99(1):53–64. doi: 10.1016/0003-2697(79)90043-5. [DOI] [PubMed] [Google Scholar]
  4. Chase T., Jr, Shaw E. Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters. Titration of the enzymes. Biochemistry. 1969 May;8(5):2212–2224. doi: 10.1021/bi00833a063. [DOI] [PubMed] [Google Scholar]
  5. Cleland W. W. The statistical analysis of enzyme kinetic data. Adv Enzymol Relat Areas Mol Biol. 1967;29:1–32. doi: 10.1002/9780470122747.ch1. [DOI] [PubMed] [Google Scholar]
  6. Leytus S. P., Peltz G. A., Liu H. Y., Cannon J. F., Peltz S. W., Livingston D. C., Brocklehurst J. R., Mangel W. F. A quantitative assay for the activation of plasminogen by transformed cells in situ and by urokinase. Biochemistry. 1981 Jul 21;20(15):4307–4314. doi: 10.1021/bi00518a011. [DOI] [PubMed] [Google Scholar]
  7. Morita T., Kato H., Iwanaga S., Takada K., Kimura T. New fluorogenic substrates for alpha-thrombin, factor Xa, kallikreins, and urokinase. J Biochem. 1977 Nov;82(5):1495–1498. doi: 10.1093/oxfordjournals.jbchem.a131840. [DOI] [PubMed] [Google Scholar]
  8. Nieuwenhuizen W., Wijngaards G., Groeneveld E. Flourogenic peptide amide substrates for the estimation of plasminogen activators and plasmin. Anal Biochem. 1977 Nov;83(1):143–148. doi: 10.1016/0003-2697(77)90519-x. [DOI] [PubMed] [Google Scholar]
  9. Pierzchala P. A., Dorn C. P., Zimmerman M. A new fluorogenic substrate for plasmin. Biochem J. 1979 Dec 1;183(3):555–559. doi: 10.1042/bj1830555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Pochron S. P., Mitchell G. A., Albareda I., Huseby R. M., Gargiulo R. J. A fluorescent substrate assay for plasminogen. Thromb Res. 1978 Nov;13(5):733–739. doi: 10.1016/0049-3848(78)90179-2. [DOI] [PubMed] [Google Scholar]
  11. Smith R. E., Bissell E. R., Mitchell A. R., Pearson K. W. Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethylcoumarin. Thromb Res. 1980 Feb 1;17(3-4):393–402. doi: 10.1016/0049-3848(80)90074-2. [DOI] [PubMed] [Google Scholar]
  12. Wong S. C., Shaw E. Inactivation of trypsin-like proteases by active-site-directed sulfonylation. Ability of the departing group to confer selectivity. Arch Biochem Biophys. 1976 Sep;176(1):113–118. doi: 10.1016/0003-9861(76)90146-6. [DOI] [PubMed] [Google Scholar]
  13. Zimmerman M., Ashe B., Yurewicz E. C., Patel G. Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates. Anal Biochem. 1977 Mar;78(1):47–51. doi: 10.1016/0003-2697(77)90006-9. [DOI] [PubMed] [Google Scholar]
  14. Zimmerman M., Yurewicz E., Patel G. A new fluorogenic substrate for chymotrypsin. Anal Biochem. 1976 Jan;70(1):258–262. doi: 10.1016/s0003-2697(76)80066-8. [DOI] [PubMed] [Google Scholar]

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