Dual effect of heparin on Fe²⁺-induced cardiolipin peroxidation: implications for peroxidation of cytochrome c oxidase bound cardiolipin

Abstract

The effect of heparin on peroxidation of cardiolipin (CL) initiated by ferrous iron was studied in vitro using detergent-solubilized CL, liposomal CL, or CL bound to isolated cytochrome c oxidase (CcO). Heparin increased both the rate and the extent of CL peroxidation for detergent-solubilized CL and for CcO-bound CL. The effect of heparin was time- and concentration-dependent as monitored by the formation of conjugated dienes or thiobarbituric acid reactive substances. The results showed great similarity between the effect of heparin and the effect of certain iron chelators, such as ADP, on phospholipid peroxidation. Heparin increased the peroxidation of CcO-bound CL only when tertiary butyl hydroperoxide was also present. The enzyme activity of the resulting CcO complex decreased 25 %, in part due to peroxidation of functionally important CL. In contrast to peroxidation of detergent-solubilized CL, peroxidation of liposomal CL was inhibited by heparin, suggesting that the effect of heparin and ferrous iron depends on their proximity to the acyl chains of CL.

Figure 1. Fe²⁺ initiated peroxidation of detergent-solubilized cardiolipin as a function of heparin concentration (upper panel) and time (lower panel)

Upper Panel: Cardiolipin (134 μM) was solubilized in 1 mL of 50 mM Tris-SO₄ buffer pH 7.4, containing of 2 mM of dodecyl maltoside. Different concentrations of heparin were added and peroxidation was started by addition of 50 μM Fe²⁺. Spectra were taken after 30 min. of incubation at room temperature: (1) CL; (2) CL + 100 μM heparin; (3) CL + Fe; (4) CL + Fe + 25 μM heparin; (5) CL + Fe + 50 μM heparin; (6) CL + Fe + 70 μM heparin; (7) CL + Fe + 100 μM heparin; (8) CL + Fe + 140 μM heparin; (9) CL + F e + 150 μM heparin. Lower Panel: Peroxidation was initiated by addition of 50 μM of Fe²⁺ or 50 μM of Fe³⁺.Concentration of heparin was 0 μM (curve 1) and 100 μM (curve 2 and curve 3 ).

Figure 2. Rate of oxidation of Fe²⁺ (upper panel) and the effect of ferric iron on detergent-solubilized cardiolipin in the presence of heparin (lower panel)

Upper panel: Ferrous iron was added to 1) dodecyl maltoside-solubilized CL with heparin (black filled squares) and without heparin (empty s quares); 2) CL liposomes with heparin (black filled circles) and without heparin (empty circles); and 3) dodecyl maltoside -solubilized CcO (empty diamonds are for both measurements with or without heparin). The initial concentration of Fe²⁺ was 50 μM; heparin was 100 μM and CcO was 5 μM. Absorbance at 515 nm was measured immediately after addition of 2.5 mM 1,10-phenanthroline. To determine the absorbance, which reflects 100% amount of Fe²⁺, 1,10-phenantroline was added in control sample before addition of ferrous ions. Lower panel: Cardiolipin (67 μM) was solubilized in 1 mL of 50 mM Tris-SO₄ buffer pH 7.4 containing 1 mg/mL of dodecyl maltoside. Concentration of heparin was 100 μM (spectrum 1). Reaction was initiated by addition of 50 μM Fe³⁺. Spectra 2, 3, 4 and 5 were taken after 0 min, 1 min, 5 min and 30 min, respectively.

Figure 3. Effect of ADP and heparin on Fe²⁺ -initiated detergent-solubilized CL peroxidation

Cardiolipin (134 μM) was solubilized in 1 mL of 50 mM Tris-SO₄ buffer pH 7.4 containing 1 mg/mL of dodecyl maltoside. Peroxidation was initiated by addition of 50 μM Fe²⁺. Spectra were taken after 30 min of incubation at room temperature: (1) CL; (2) CL+Fe; (3) CL +Fe+100 μM ADP; (4) CL +Fe+100 μM ADP+100 μM heparin (solid line); (5) CL + Fe + 100 μM heparin (dotted line).

Figure 4. Heparin inhibi ts Fe²⁺ induced peroxidation of cardiolipin in liposomes

Liposomes were made from 134 μM CL as described in Material and Method. Peroxidation was initiated by addition of 50 μM of Fe²⁺. After 30 min incubation at room temperature cardiolipin was extracted by methanol – chloroform, dried under nitrogen and dissolved in 0.5 mL of ethanol. (1) CL; (2) CL + Fe + 100 μM heparin; (3) CL + Fe

Figure 5. Silicic acid HPLC and spectral analysis of cardiolipin extracted from cytochrome c oxidase

Upper panel – silicic acid HPLC chromatogram. The numbers (I, II and III) designate the randomly chosen areas where spectra were analyzed. Lower panels ( b, c and d ) represent spectra extracted from the randomly chosen areas (I, II and III) of silicic acid HPLC. Solid line is CL extracted from 5 μM CcO; dotted line is CL extracted from 5 μM CcO incubated with 5 mM tert-BOOH and 50 μM Fe²⁺; dashed line is CL extracted from 5 μM CcO incubated with 5 mM tert-BOOH, 50 μM Fe²⁺ and 100 μM heparin.

Figure 6. Inactivation of CcO electron transport activity by tert-BOOH, ferrous ion with and without heparin

CcO (5μM) solubilized in 50 mM Tris-SO⁴ buffer, pH 7.4, containing 2 mM dodecyl maltoside and 2 mM sodium cholate was incubated for 60 min at room temperature with: (1) no additions; (2) 5 mM tert-BOOH ± 100 μM heparin ; (3) 5 mM tert-BOOH + 50 μM Fe²⁺; (4) 5 mM tert-BOOH + 50 μM Fe²⁺ + 100 μM heparin; (5) 50 μM Fe²⁺ ; (6) CcO + 50 μM Fe²⁺ + 100 μM heparin. The reaction was stopped by dilution and the activity measured spectrophotometrically with reduced cytochrome c as a substrate. These results are representative of three independent experiments. The error in determination of molecular activity was ±2.6%.