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New fluorogenic peptide substrates for plasmin.

H Kato, N Adachi, Y Ohno

    Journal of Biochemistry
    |July 1, 1980
    PubMed
    Summary
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    New fluorogenic substrates, Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA, enable specific and sensitive plasmin assays. These peptides show resistance to various proteases and streptokinase-activated plasmin, enhancing assay reliability.

    Area of Science:

    • Biochemistry
    • Enzymology
    • Protease assays

    Background:

    • Plasmin is a key enzyme in fibrinolysis.
    • Development of specific and sensitive substrates is crucial for plasmin research and diagnostics.
    • Existing substrates may lack specificity or be affected by other proteases.

    Purpose of the Study:

    • To synthesize and characterize novel fluorogenic peptide substrates for plasmin.
    • To evaluate the specificity and sensitivity of these substrates against plasmin and other proteases.
    • To assess the utility of these substrates in the presence of streptokinase-activated plasmin.

    Main Methods:

    • Synthesis of six fluorogenic peptidyl-4-methylcoumaryl-7-amides (MCA) with C-terminal lysine.
    • Assay of plasmin activity using synthesized substrates.

    Related Experiment Videos

  • Determination of kinetic parameters (Km) using Line-weaver-Burk plots.
  • Testing substrate hydrolysis by various proteases including kallikreins, urokinase, and thrombin.
  • Evaluating substrate stability against streptokinase-activated plasmin.

    • Main Results:

    • Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA identified as effective substrates for plasmin.
    • Km values for human and bovine plasmin were in the 10(-4) M range.
    • Substrates showed high specificity, with minimal hydrolysis by other tested proteases.
    • Boc-Glu-Lys-Lys-MCA and Boc-Val-Leu-Lys-MCA were resistant to streptokinase-activated plasmin.

    Conclusions:

    • Boc-Val-Leu-Lys-MCA and Boc-Glu-Lys-Lys-MCA are valuable tools for specific and sensitive plasmin assays.
    • These substrates offer improved reliability due to their resistance to common interfering proteases and streptokinase.
    • The findings support the use of these novel fluorogenic peptides in biochemical and diagnostic applications involving plasmin.