Henrik Østdal

Reaction between protein radicals and other biomolecules

The present study investigates the reactivity of bovine serum albumin (BSA) radicals towards different biomolecules (urate, linoleic acid, and a polypeptide, poly(Glu-Ala-Tyr)). The BSA radical was formed at room temperature through a direct protein-to-protein radical transfer from H(2)O(2)-activated immobilized horseradish peroxidase (im-HRP). Subsequently, each of the three different biomolecules was separately added to the BSA radicals, after removal of im-HRP by centrifugation. Electron spin resonance (ESR) spectroscopy showed that all three biomolecules quenched the BSA radicals. Subsequent analysis showed a decrease in the concentration of urate upon reaction with the BSA radical, while the BSA radical in the presence of poly(Glu-Ala-Tyr) resulted in increased formation of the characteristic protein oxidation product, dityrosine. Reaction between the BSA radical and a linoleic acid oil-in-water emulsion resulted in additional formation of lipid hydroperoxides and conjugated dienes. The results clearly show that protein radicals have to be considered as dynamic species during oxidative processes in biological systems and that protein radicals should not be considered as end-products, but rather as reactive intermediates during oxidative processes in biological systems hereby supporting recent data.

Formation of Long-Lived Protein Radicals in the Reaction Between H2O2-Activated Metmyoglobin and Other Proteins

Free radicals formed during the reaction of H2O2 and metmyoglobin in the presence of bovine serum albumin (BSA) were investigated using freeze quench and spin-trap ESR spectroscopy. Increasing concentrations of BSA (0-300 microM) resulted in drastic changes in the characteristic freeze quench ESR signal of H2O2-activated metmyoglobin (perferryl protein radical) under physiological conditions (pH = 7.4; I = 0.16). The radical species formed during reaction of 100 microM H2O2, 100 microM metmyoglobin, and 200 microM BSA have half-lives of approximately 13 min at 25 degrees C, in contrast to the perferryl protein radical that has a half-life of approximately 28 s at 25 degrees C. The radical species formed in the presence of BSA were reactive towards ascorbate, glutathione, cysteine, and tyrosine. Substitution of BSA with defatted BSA, gamma-globulin or beta-lactoglobulin also resulted in formation of long-lived free radical species (half-lives: 13-18 min); however, the ability to form these was dependent of the specific protein and decreased in the following order: BSA > defatted BSA > gamma-globulin > beta-lactoglobulin. The spin-trap alpha-phenyl-tert-butylnitrone (PBN) showed the presence of transient protein radical species formed in the reaction between MMb, H2O2, and BSA. Transient radical species that could be proposed as intermediates in the formation of the long-lived protein radicals detected by freeze-quench ESR. Dityrosine was formed in the reaction between MMb, H2O2, and BSA, showing the involvement of tyrosine residues in the present reaction. The described chemical interaction between H2O2-activated myoglobin and other proteins have major consequences on future interpretations of the significance of the perferryl protein radical in biological systems where proteins are abundant.

Reduction of ferrylmyoglobin by beta-lactoglobulin.

Reduction of iron (IV) in ferrylmyoglobin in the presence of beta-lactoglobulin in aqueous solution is the result of two parallel reactions: (i) a so-called autoreduction, and (ii) reduction by beta-lactoglobulin in a second-order-reaction resulting in bityrosine formation in beta- lactoglobulin. In the pH-region investigated (5.4-7.4), the rate of reduction increased for both reactions with decreasing pH. The second order-reaction had for non-denatured beta-lactoglobulin the activation parameters: delta H* = 45 kJ.mol-1 and delta S not equal to = -93 J.mol-1.K-1 at pH = 7.0 and ionic strength 0.16 (NaCl). Reduction of ferrylmyoglobin by beta-lactoglobulin denatured by heat (86 degrees C for 3 min) or by hydrostatic pressure (300 MPa for 15 min) resulted in formation of higher molecular weight species as detected by size-exclusion chromatography and by SDS-PAGE. No molecular weight changes were observed for reduction of ferrylmyoglobin by native beta-lactoglobulin. Detection of bityrosine in the native beta-lactoglobulin fraction after oxidation with ferrylmyoglobin indicated intra-molecular bityrosine formation. In heat-denatured beta-lactoglobulin bityrosine formation could be of intra-molecular and/or of inter-molecular origin, the latter being confirmed by size-exclusion chromatography.

The effect of pH on the oxidation of bovine serum albumin by hypervalent myoglobin species.

Bovine serum albumin (BSA) was used as a probe for the oxidation of proteins by hypervalent myoglobin species in solutions with pH from 5.3 to 7.7. The reaction between perferrylmyoglobin, *MbFe(IV)=O, and BSA was studied by activating metmyoglobin with equimolar amounts of hydrogen peroxide in the presence of BSA. A minor pH dependence was observed as judged from the formation of BSA-centered radicals, which were monitored at room temperature by electron spin resonance spectroscopy, and the formation of dityrosine. The reaction between ferrylmyoglobin, MbFe(IV)=O, and BSA was pH-dependent. BSA-centered radicals and dityrosine were formed in low levels at neutral pH and increased at low pH to the same levels as observed in the reaction of *MbFe(IV)=O with BSA. The present results demonstrate that protein-centered radicals can be formed from the non-radical MbFe(IV)=O under mildly acidic conditions, and this should be taken into account when considering oxidation in cellular compartments of low pH and in meat-related products.

Lipases in Wheat Breadmaking: Analysis and Functional Effects of Lipid Reaction Products

The baking activity of two different lipases was evaluated by a microbaking test on a 10 g flour basis, and the altered lipid composition of lipase-treated wheat lipids was quantitated. To identify and quantitate the various lipid classes, pure glycolipids and phospholipids were isolated from a wheat flour lipid extract by a silica gel batch procedure and silica gel column chromatography. These reference compounds were used to establish a high-performance liquid chromatographic method with evaporative light scattering detection, which was able to separate all of the wheat lipid classes and lipase reaction products. Wheat lipids, dough lipids, and dough lipids after lipase addition were quantitated using cholesterol as an internal standard. Especially digalactosyl diglycerides (-0.9 mmol/kg flour), monogalactosyl diglycerides (-0.4 mmol/kg), and N-acyl-phosphatidyl ethanolamine (-0.3 mmol/kg) were hydrolyzed, and a concomitant formation of digalactosyl monoglycerides (+0.6 mmol/kg), monogalactosyl monoglycerides (+0.6 mmol/kg), and N-acyl-lysophosphatidyl ethanolamine (+0.5 mmol/kg) was found. The lipase-induced changes of the lipid fraction caused increases in bread volume of 56-58%, depending on the type and concentration of the added lipase. The current results confirm the important relationship between the lipid fraction composition and the baking performance of flour.

Phenolic Antioxidant Scavenging of Myosin Radicals Generated by Hypervalent Myoglobin

The scavenging activity of extracts of green tea (GTE), white grape (WGE), and rosemary (RE), all plant material with high phenolic content, and of the phenolic compounds 4-methylcatechol (4-MC), (+)-catechin, and carnosic acid toward long-lived myosin radicals generated by reaction with H2O2-activated myoglobin at room temperature (pH 7.5, I=1.0) was investigated by freeze-quench ESR spectroscopy. Myosin radicals were generated by incubating 16 μM myosin, 800 μM metmyoglobin, and 800 μM H2O2 for 10 min, and the phenolic extracts were subsequently added (1% (w/w) phenolic compounds relative to myosin). GTE was able to scavenge myosin radicals and reduce the radical intensity by 65%. Furthermore, a low concentration of 4-MC (33 μM) was found to increase the radical concentration when added to the myosin radicals, whereas a higher concentration of 4-MC and catechin (330 μM) was found to scavenge myosin radicals and reduce the overall radical concentration by ∼65%.

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (·MbFe(IV)=O) and/or ferrylmyoglobin (MbFe(IV)=O). Reduction of MbFe(IV)=O showed first-order kinetics for a 1–2 times stoichiometric excess of H2O2 to MMb while a 3–10 times stoichiometric excess of H2O2 resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H2O2 and BSA were influenced by [H2O2] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H2O2. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H2O2 to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H2O2 and the hyper-valent iron-center of H2O2-activated MMb.

Reconstitution baking tests with defatted wheat flour are suitable for determining the functional effects of lipase-treated wheat lipids.

A microscale reconstitution baking test, using wheat flour defatted with 2-propanol at 20 °C, was established to determine the functional effects of lipids isolated from lipase-treated wheat dough. Proper selection of solvent and extraction temperature was of major importance to maintain the functionality of defatted flour. Dough and gluten from flour defatted with water-saturated 1-butanol (WSB; extracted at 20 °C) and 2-propanol (extracted at 75 °C) had inferior extensibility and loaf volume compared to control flour extracted with 2-propanol at 20 °C. Quantitation of gluten proteins showed that defatting with WSB (20 °C) or 2-propanol (75 °C) decreased the gliadin and increased the glutenin content. Possible reasons were thiol-disulfide interchange reactions, caused either by heat (2-propanol, 75 °C) or by the solvent WSB, which affected gluten proteins. Confocal laser scanning microscopy showed that regular, interconnected gluten structures were only present in dough from flour defatted with 2-propanol at 20 °C.