The Determination of the Heparin Neutralising Capacity of Protamine Using Acridine Orange Fluorescence

Haydn E Edwards; Suppiah Navaratnam; John C Allen; Glyn O Phillips

doi:10.1055/s-0038-1650028

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1980; 43(02): 108-111
DOI: 10.1055/s-0038-1650028

Original Article

Schattauer GmbH Stuttgart

The Determination of the Heparin Neutralising Capacity of Protamine Using Acridine Orange Fluorescence

Authors

Haydn E Edwards

The Research Division of the School of Natural Science, The North East Wales Institute, Kelsterton College, Clwyd, Wales, U. K.
Suppiah Navaratnam

The Research Division of the School of Natural Science, The North East Wales Institute, Kelsterton College, Clwyd, Wales, U. K.
John C Allen

The Research Division of the School of Natural Science, The North East Wales Institute, Kelsterton College, Clwyd, Wales, U. K.
Glyn O Phillips

The Research Division of the School of Natural Science, The North East Wales Institute, Kelsterton College, Clwyd, Wales, U. K.

Abstract

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Summary

A new method to estimate the heparin neutralising capacity of protamine has been investigated. The technique has been applied to two commercial heparin preparations tested against the W.H.O. 1st International Reference preparation of protamine.

The technique is based on the fluorescence of acridine orange dye and the selective binding of heparin with protamine. A titration procedure has been devised and the two variations of the assay are compared and evaluated. The titration is rapid, accurate and convenient.

Keywords

Heparin - Protamine - Acridine orange - Fluorescence - Titration

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References

References
1 Graham DT, Pomeroy AR, Smythe DB. Measurement of the heparin neutralising capacity of protamine. Thromb Haemostas (Stuttg.) 1979; 41: 583-589
2 Cundall RB, Phillips GO, Rowlands DP. A spectrofluorimetric procedure for the assay of carrageenan. Analyst 1973; 98: 857-862
3 Diakun GP, Edwards HE, Allen JC, Phillips GO. Analysis of Glycosaminoglycans in urine by using acridine orange fluorescence. Biochem J 1978; 175: 573-577
4 Lamm ME, Neville DM. The dimer spectrum of acridine orange hydrochloride. J. Physical Chem 1965; 69: 3872-3877
5 Stone AL, Bradley DF. Aggregation of acridine orange to polyanions: the stacking tendencies of deoxyribonucleic acids. J American Chem Soc 1961; 83: 3627-3634
6 Rowlands DP. Investigation of dye macromolecule complexes using spectrofluorimetric techniques. Ph D Thesis 1974: University of Salford
7 Cundall RB, Lawton JB, Murray D, Phillips GO. The role of co-operativity in determining the binding behaviour of hetero-charged polyanions with metachromatic dyes. Makromol Chem 1979; 180: 383-388
8 Edwards HE, Diakun GP, Davies JV, Allen JC, Phillips GO. The anticoagulant activity of heparin – a fluorescence and pulse radiolysis study. Biochem Biophys Res Comm 1978; 85: 1602-1609
9 Lindahl U, Backstrom G, Hook M, Thunberg L, Fransson LA, Linker A. Structure of the Antithrombin-binding site in Heparin. Proc Nat Acad Sci USA 1979; 76: 3198-3202
10 Diakun GP, Edwards HE, Allen JC, Phillips GO, Cundall RB. A simple purpose built fluorimeter for the titrimetric assay of glycosaminoglycans. Anal Biochem 1979; 94: 378-382
11 Bangham DR, Woodward PM. A collaborative study of heparins from different sources. Bull WHO 1970; 42: 129-149