Dose-response Curves in the Fibrin Plate Assay Fibrinolytic Activity of Proteases

Summary

In the fibrin plate assay different types of relationships between the dose of applied proteolytic enzyme and the response have been previously reported. This study was undertaken to determine whether a generally valid relationship might exist.

Trypsin, chymotrypsin, papain, the plasminogen activator urokinase and all of the microbial proteases investigated, including brinase gave a linear relationship between the logarithm of the enzyme concentration and the diameter of the circular lysed zone. A similar linearity of dose-response curves has frequently been found by investigators who used enzyme plate assays with substrates different from fibrin incorporated in an agar gel. Consequently, it seems that this linearity of dose-response curves is generally valid for the fibrin plate assay as well as for other enzyme plate bioassays.

Both human plasmin and porcine tissue activator of plasminogen showed deviations from linearity of semi-logarithmic dose-response curves in the fibrin plate assay.

• References

• 1 Arnon R. 1970. Papain. In: Colowick S. P, Kaplan N.O. (ed.), Methods in Enzymology XIX. Academic Press; London: 226.
• 2 Astrup T, Alkjaersig N. 1952; Estimation of proteolytic enzymes by means of their fibrinolytic activity. Archives Biochemistry and Biophysics 37: 99.
• 3 Astrup T, Kok P. 1970. Assay and preparation of a tissue activator. In Colowick S. P, Kaplan N. O. (ed.), Methods in Enzymology XIX. Academic Press; London: 821.
• 4 Astrup T, Mullertz S. 1952; The fibrin plate method for estimating fibrinolytic activity. Archives of Biochemistry and Biophysics 40: 346.
• 5 Binder B, Zarrabi Z. 1972; Zur Frage der direkten Aktivatorwirkung von Streptokinase auf Plasminogen. Wiener Medizinische Wochenschrift 122: 104.
• 6 Brakman P. 1967. Fibrinolysis, a standardized fibrin plate method and a fibrinolytic assay of plasminogen. Scheltema and Holkema; Amsterdam.:
• 7 Carter D. V, Sykes G. 1961; The assay of enzyme activity by the plate diffusion technique. Journal of Pharmacy and Pharmacology 13: 195.
• 8 Cooper KE. 1963. The theory of antibiotic inhibition zones. In: Kavanagh F. (ed.), Analytical Microbiology. Academic Press; London: 1.
• 9 Crank J. 1970. The Mathematics of Diffusion. Oxford University Press; London: 27.
• 10 Fossum K. 1970 Proteolytic enzymes and biological inhibitors. Acta Pathologica et Microbiologica Scandinavica. 78, 350, 605, 741, 755.
• 11 Habermann E, Hardt KL. 1972; A sensitive and specific plate test for the quantitation of phospholipases. Analytical Biochemistry 50: 163.
• 12 Haverkate F. 1972; A simple device for measuring diameters of fibrinolysis zones in fibrin plates. Haemostasis 01: 55.
• 13 Heberlein PJ, Barnhart MI. 1971. The purification of profibrinolysin and fibrinolysin. In: Bang NU, Beller FK, Deutsch E, Mammen EF. (ed.), Thrombosis and Bleeding Disorders. Academic Press, London and Georg Thieme Verlag; Stuttgart: 336.
• 14 Holmstrom B. 1965; Streptokinase assay on large agar diffusion plates. Acta Chemica Scandinavica 19: 1549.
• 15 Jarvis AW, Lawrence RC. 1969; A micro-method for the quantitative determination of DNAse. Canadian Journal of Biochemistry 47: 673.
• 16 Kok P, Astrup T. 1969; Isolation and purification of a tissue plasminogen activator and its comparison with urokinase. Biochemistry 08: 79.
• 17 Lachica R. V. F, Hoeprich PD, Franti CE. 1972; Convenient assay for staphylococcal nuclease by the metachromatic well-agar-diffusion technique. Applied Microbiology 24: 920.
• 18 Lawrence BC, Fryer TF, Beiter B. 1967; Rapid method for the quantitative estimation of microbial lipases. Nature 213: 1264.
• 19 Mestecky J, Kraus FW, Hurst DC, Voigt SA. 1969; A simple quantitative method for a-amylase determination. Analytical Biochemistry 30: 190.
• 20 Moers AMJ. 1962 De meting van fibrinolyse. Thesis. Amsterdam.:
• 21 Mullertz S. 1952; The estimation of plasmin and trypsin by means of the plate method. Acta Physiologica Scandinavica 25: 93.
• 22 Oberzill W. 1967; Mikrobiologische Analytik. Verlag Hans Carl, Nürnberg 35-379.
• 23 Osserman EF, Lawlor DP. 1966; Serum and urinary lysozome (muramidase) in monocytic and monomyelocytic leukemia. Journal of Experimental Medicine 124: 921.
• 24 Permin PM. 1947; Properties of the fibrinokinase-fibrinolysin system. Nature 160: 571.
• 25 Samloff IM, Kleinman MS. 1969; A radial diffusion assay for pepsinogen and pepsin. Gastroenterology 56: 30.
• 26 Schill WB, Schumacher GFB. 1972; Radial diffusion in gel for micro determination of enzymes I. Muramidases, a-amylase, DNAse I, RNAse, acid phosphatase and alkaline phosphatase. Analytical Biochemistry 46: 502.
• 27 Schumacher GFB, Schill WB. 1972; Radial diffusion in gel for micro determination of enzymes II. Plasminogen activator, elastase and non specific proteases. Analytical Biochemistry 48: 9.
• 28 Sgouris JT, Inman JK, McCall KB, Hyndman LA, Anderson HD. 1960; The preparation of human profibrinolysin (plasmin). Vox Sanguinis 05: 357.
• 29 Thorsen S, Glas-Greenwalt P, Astrup T. 1972; Differences in the binding to fibrin of urokinase and tissue plasminogen activator. Thrombosis et Diathesis Haemorrhagica 28: 65.
• 30 Walton PL. 1966; An improved fibrin plate method for the assay of plasminogen activator. Clinica chimica Acta 13: 680.