Effect of chemically modified heparins, and of heparin fragments, on Fe(I1)-catalyzed peroxidation of linolenic acid. MARION A. ROSS, WILLIAM F. LONG, ...
2 16s Biochemical SocietyTransactions ( 1 992) 20 Effect of chemically modified heparins, and of heparin fragments, on Fe(I1)-catalyzed peroxidation of linolenic acid.
0.12
MARION A. ROSS, WILLIAM F. LONG, FRANK B. WILLIAMSON and COLIN F.MOFFAT
0.10
Department of Molccular and Cell Biology, University of Abcrdeen, Marischal College, ABERDEEN AB9 IAS, Scotland. Heparins possess a variety of anionic groups that allow the molecules to interact with a range of biologically relevant micro- and macro-cations. These molecules might therefore influence the activity of radicals by modulating the availability or reactivity of those transition metal ions with putative roles in first-chain initiation of lipid peroxidation and the ability to accelerate decomposition of lipid peroxides. The present transaction uses a simple in virro model to investigate the possible effects of heparin on lipid peroxidation. The source, preparation and properties of the heparin have been described previously [1.2]. Chemically modified forms of heparin were prepared and characterized as described by Grant et a/.[2]. Heparin fragments were generated by the action of heparinase I as described by Moffat [3]. In Fig. 2, pools 4, 5 and 6 refer to fractions containing octa-, tetra- and di-saccharides respectively, pool 3 to a fraction containing non-sulphated N-acetylated fragments. and pools 2 and 1 to fractions containing fragments of progressively higher degrees of polymerization. y -Linolenic acid (6,9,12-octadecamenoic acid) and polyoxyethylene ether a-1 (adodecyl-a-hydroxypoly (oxy- 1.2-ethanediyl)) were from Sigma Chemical Co., Poole. U.K. Ferrous ammonium sulphate (Specpure grade) was from Johnson Matthey. Royston, U.K. Assay of an early stage in linolenic acid peroxidation, the formation of u.v.absorbing conjugated dienes, was essentially by the method of Knight & Voorhees [4]. Na+ forms of the polymers (at final concentrations of 1 mmol.dmd with regard to the hydrated dimer in the case of the chemically modified polymers, and 0.1 mm0l.dm-3 in the case of the heparin fragments) and polyoxyethylene ether o 1 (1% w/v) were mixed at 37OC in a spectrophotometer cell and allowed to equilibrate for 10 min before addition of y-linolenic acid
0.12
0.08
0.04
0.00
Fig. 1 Effect of chemically modified heparins on conjugate diene production Modification of heparin used: (a)no heparin present; (b) de-Nsulphonation, de-0-sulphation; (c) de-N-sulphonation, de-0sulphation, N-acetylation; ( d ) de-N-sulphonation; (e) carboxyl reduction; (f) de-N-sulphonation, de-0-sulphation, re-Nsulphonation; (g) de-N-sulphonation,N-acetylation; (h) unmodified heparin.
E
C
0.08
m
m
hl
%
0.06
8
C 0 0
p
0.04
9 0.02
0.00
Fig. 2 Effect of heparin fragments on conjugate diene production Pool of heparin fragments used: (a)no heparin present; (6)6; (c) 5; (d)4; (e) 3; v) 2; (g) 1; (h) unfractionated heparin.
in 95% (v/v) ethanol (0.3 m m ~ l . d m -and ~ ) incubation for a further 5 min. Ferrous ammonium sulphate (2.5 x 1 0 5 mol.dm-3; deoxygenated) was then added, and absorbance measured at 233 nm after 10 min. Reagents were made up in deionized distilled water and had the above noted concentrationsin a final reaction mix of 3 cm3. Heparin, in a concentration-dependent manner, inhibits the increase in U.V. absorption due to conjugate diene production that follows addition of Fe(I1) to y-linolenic acid in the presence of molecular oxygen. Heparin which had been re-N-sulphonated following removal of N-sulphonates and 0-sulphates, and heparin in which iduronate residues had been reduced to idose residues were largely unaffected in their activity (Fig. I). Removal of both Nsulphonates and O-sulphates greatly reduced the effectiveness of the heparin. Replacement of N-sulphonates by N-acetyl groups slightly potentiated the action of heparin. Analysis of the effects of fragments generated by the action of heparinase on heparin (Fig. 2) showed that depolymerization reduced the effectiveness of the heparin. It is suggested that heparin acts as an antioxidant by binding and sequestering Fe(I1) ions. We thank the Scottish Home and Health Department for a grant supporting this work. 1. Grant, D., Long, W. F. &Williamson, F. B. (1987) Biochem. J. 244, 143-149 2. Grant, D., Long, W. F., Moffat, C. F. & Williamson, F. B. (1989) Biochem. J. 261, 1035-1038 3. Moffat, C.F. (1987) PhD thesis, University of Aberdeen 4. Knight, J. A. & Voorhees, R. P. (1990) Ann. Clin. Lab. Sci. 20, 341-352
