Oxidative denaturation

Enzyme Sta.bihty, Loss of enzyme-catalytic activity may be caused by physical denaturation, eg, high temperature, drying/freezing, etc or by chemical denaturation, eg, acidic or alkaline hydrolysis, proteolysis, oxidation, denaturants such as surfactants or solvents, etc. pH has a strong influence on enzyme stabiHty, and must be adjusted to a range suitable for the particular enzyme. If the enzyme is not sufficiendy stable in aqueous solution, it can be stabilized by certain additives a comprehensive treatment with additional examples is available (27). 

The shaking of protein solutions may lead to aggregation and precipitation as a result of several mechanisms, such as air oxidation, denaturation at the interface, adsorption to the vessel, or mechanical stress. These possibilities were systematically examined for solutions of human fibroblast interferon [50]. In this example, mechanical stress was identified as the causative factor in the inactivation. The proposed mechanism of inactivation by mechanical stress was through orientation of the asymmetrical protein in the... 

The most important product of the hexose monophosphate pathway is reduced nicotinamide-adenine dinucleotide phosphate (NADPH). Another important function of this pathway is to provide ribose for nucleic acid synthesis. In the red blood cell, NADPH is a major reducing agent and serves as a cofactor in the reduction of oxidized glutathione, thereby protecting the cell against oxidative attack. In the syndromes associated with dysfunction of the hexose monophosphate pathway and glutathione metabolism and synthesis, oxidative denaturation of hemoglobin is the major contributor to the hemolytic process. 

The presence of precipitates of oxidized, denatured hemoglobin (Heinz bodies) helps distinguish the hemolytic anemia caused by of G6PD deficiency from that caused by pyruvate kinase deficiency. 

The aim is to extract protein molecules as pure as possible. Detergents generally help membrane proteins to dissolve and separate from lipids. Reductants are used to reduce disulfide bonds or prevent oxidation. Denaturing agents alter ionic strength... 

Oxidized, denatured hemoglobin forms aggregates, which can become attached to the inner surface of the red cell, known as Heinz bodies. This leads to damage to the red cell, which may result in direct destruction of the cell, which can be shown in vitro, or removal from the circulation by the spleen in vivo. When caused by Fava beans, the syndrome is known as Favism. As the deficient enzyme (glucose-6-phosphate dehydrogenase) is intrinsic to the red cell, exposure of such cells in vitro to suitable drugs will lead to cell damage and death. 

The major hemoglobin variant in subjects with this condition is Hb-H. Hb-H consists solely of four jff chains and its gross structure is (JH). The variant is heat labile, rather acid resistant (B34), unusually susceptible to oxidative denaturation, and has a 10- to 12-fold increase in affinity for molecular oxygen (B27). The proportion of Hb-H in cases with Hb-H disease ranges from 2 to 40% the accuracy of these data is questionable because of diflBculties in quantitating this unstable variant. 

The first indication that electron transfer involving FeS-A/FeS-B may follow a branched pathway was reported by Golbeck and Warden in 1982. It was found that treating a PS-I complex with urea plus ferricyanide induced oxidative denaturation ofthe Fe-S centers, i.e., conversion. The iron-sulfur... 

Electrophoretic assays may be used to characterize the purity and homogeneity of biopharmaceuticals, based mainly on an ability to differentiate chemical and molecular changes in the compound as a result of oxidation, denaturation, aggregation, and deamidation [11]. Well-established techniques include isoelectric focusing (lEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whilst high-performance capillary electrophoresis (HPCE), utilizing a variety of detection systems, may also be used for routine applications. 

Pacifici, R.E., Salo, D.C, and Davies, K.J.A. (1989) Macroxyproteinase (MOP) a 670-kDa proteinase complex that degrades oxidatively denatured proteins in red blood cells. Free Radical Biol. Med. 7 521-536. 

Marcillat. O, Zhang, Y, Lin, S.W., Davies, K.J.A., Mitochondria Contain a Proteolytic System Which can Recognize and Degrade Oxidatively-Denatured Proteins, Biochemical Tournal. 254, 677 (1988). 

Fx Ffi and Fa can be oxidatively denatured and reconstituted from FeCls and Na2S,... 

The major difficulty with assigning a cluster identity for Fx is that the Fa and Fb clusters, which constitute 8 out of the 12 iron atoms in the Photosystem I core complex, contribute most of the backscattering to the EXAFS spectrum. This problem can be circumvented by using the newly isolated Photosystem I core protein incorporating the components P700, Aq, (Ai by inference) and Fx, but devoid of Fa and Fb. In this preparation, the X-ray K-edge spectrum was found to be similar to that of four-coordinate [4Fe-4S] clusters and unlike six-coordinate iron complexes that are present in heat-denatured Photosystem I or oxidatively denatured ferredoxin. This indicates that the 4 iron atoms in the Photosystem I core protein are most certainly in the form of an intact iron-sulfur complex. The k-space spectrum of Fx (Fig. 1) can be simulated by assuming... 

Our current work with the Photosystem I core protein reconfirms that the Fx iron-sulfur cluster can be oxidatively converted to zero-valence sulfur by treatment with 3 M urea and 5 mM potassium ferricyanide [13,14]. Under these conditions, the P700 flash-induced optical transient has a lifetime of 5 is due to the relaxation of the P700 triplet state, indicating that the P700 Aq primary charge separation and recombination process has remained intact (Fig. 3a,b). We have now found that the Fx iron-sulfur cluster can be reconstituted by addition of ferrous iron, sodium sulfide, and 6-mercaptoethanol to the oxidatively-denatured Photosystem I core protein [see refs. 15 16 for methodology employed]. After incubation for 24 hr, the 5 is optical transient becomes replaced with a 1.2-ms optical transient that is characteristic of the P7(X)+-Fx backreaction (Fig. 3c). 

Fig. 3. Flash-induced absorption changes in the Photosystem I core protein. (A) Absorption transient in the native-Fx Photosystem I core protein. (B) Absorption transient in the apo-Fx Photosystem core protein prepared by oxidative denaturation with 3 M urea and 5 mM potassium ferricyanide. Studies at a faster digitizing rate showed that the P700 absorption transient had approximately the same magnitude with a half-time of 5 xs (not shown). (C) Absorption transient in the reconstituted-Fx Photosystem I core protein prepared by incubation of apo-Fx with inorganic iron and sulfide in the presence of 6-mercaptoethanol. All measurements were performed at 5 jxg Chl/ml in 50 mM Tris buffer, pH 8.3, containing 1.7 mM ascorbate and 0.033 mM DCPIP.

The ESR spectra of the native-Fx, apo-Fx and reconstituted-Fx Photosystem I core proteins are shown in Fig. 4. The light-induced spectrum of native-Fx (Fig. 4A) has resonances at g = 2.05, 1.86 and 1.78, and is similar to that described previously [7]. Figure 4B shows that after oxidative denaturation, little or no light-inducible Fx is present, in agreement with the optical determination of apo-Fx shown in Figure 3B. After reconstitution with inorganic iron and sulfide in the presence of 6-mercaptoethanol, the characteristic ESR spectrum of Fx is recovered (Fig. 4C). 

We found that the oxidatively-denatured Fa/Fb iron-sulfur clusters can also be reconstituted by incubating the apoprotein with FeCls and Na2S in the presence of 6-mercaptoethanol. The ESR spectra of the native Fa/Fb protein, the Fa/Fb apoprotein and the reconstituted Fa/Fb protein are shown in Fig. 5. The chemically-reduced spectmm of... 

Fig. 5. ESR spectra of the chemically-reduced Fa/Fb proteins. (A) spectrum of the native-FA/Ffi protein. (B) spectrum of the Fa/Fb apoprotein prepared by oxidative denaturation with 3 M urea and 5 mM K3(FeCN)6. (C) spectrum of the reconstituted-FA/FB protein prepared by reconstitution of the Fa/Fb apoprotein with FeCls and Na2S in the presence of 6-mercaptoethanol. The samples were incubated 4 min with sodium dithionite and 0.033 mM methyl viologen in 0.1 M glycine, pH 10. Spectrometer conditions temperature, 16 K microwave power, 20 mW microwave frequency, 9.128 GHz receiver gain, 5.0 x 10 modulation amplitude, 10 G at 100 kHz.

Many biochemical reactions can be induced by temperature increase in foods Maillard reactions, vitamin degradation, fat oxidation, denaturation of thermally unstable proteins (resulting in variation of solubility or of the germinating power of grains, for example), enzyme reactions (which can either be promoted or inhibited), and so on. Some of these biochemical reactions generate components suitable, for example, for their sensory properties (flavor development) others may be more or less undesirable for nutritional or potential toxicity reasons (vitamin losses, changes in color, taste or aroma, formation of toxic compounds). All the reactions are linked to the simultaneous evolution of product composition, temperature and water content (or chemical potential, or water activity), these factors varying diflferently from one point to another, from the center to the surface of the products. 

Maintenance of the active reducing potential via NADPH to protect hemoglobin and enzyme proteins from oxidative denaturation... 

Inchviduals with this condition have good health until a haemolytic crisis is precipitated by oxidative stress, such as bacterial infections, antibiotics, anrimalarial drugs, certain dyes, or the ingestion of fava beans (similar to broad beans). A blood film from a patient who has this condition will often show the presence of Heinz boches inside the RBCs, which result from the precipitation of oxidized, denatured haemoglobin. 

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