RBC Metabolism Notes (Part 1) : Stages of oxidative denaturation of Hemoglobin

OXIDATIVE DENATURATION OF HEMOGLOBIN: IT’S REVERSIBILITY AND PREVENTION

INTRODUCTION

• Oxyhemoglobin in solution gradually undergoes autoxidation, becoming methemoglobin (HbFe⁺⁺⁺).
• The rate of oxidation is enhanced by conditions such as increased temperature, decreased pH and presence of organic phosphate and of metal ions, and partial oxygenation of hemoglobin. To bind oxygen reversibly, however, the iron in the heme moiety must be maintained in the reduced (ferrous Fe⁺⁺) state, despite exposure to a variety of endogenous and exogenous oxidizing agents.
• The red cells maintain several metabolic pathways to prevent the action of these oxidizing agents and to reduce the hemoglobin iron if it becomes oxidized. Under certain circumstances, these mechanisms fail and hemoglobin becomes non-functional. At times, hemolytic anemia supervenes as well.
• These abnormalities are particularly likely to occur
• if the red cell is exposed to certain oxidant drugs or toxins,
• if the intrinsic protective mechanisms of the cell are defective or
• if genetic abnormalities of the hemoglobin molecule are affecting globin stability or the heme crevice.

  STAGES IN OXIDATIVE DENATURATION OF HEMOGLOBIN

• The oxidation of hemoglobin occurs in a stepwise fashion from fully reduced hemoglobin to fully oxidized hemoglobin. Intermediate forms are called valence hybrids.
• In deoxyhemoglobin, the heme iron is in the "high spin" ferrous state, in which six electrons are in the outer shell, four of which are unpaired. When oxygen is added, one of these electrons is partially transferred to the bound oxygen.
• Usually, when oxygen is given up, oxyhemoglobin dissociates into partially deoxygenated hemoglobin and molecular oxygen:

 Hb(O₂)₄ -------> Hb(O₂)₃ + O₂

Methemoglobin

• A superoxide anion rather than molecular oxygen may dissociate, however, thus oxidizing the Fe to the ferric state, producing methemoglobin:
• HbFe₂ + O₂--------->   HbFe³⁺O^2----------->   HbFe³⁺ + O^2-
• This type of dissociation is particularly likely if water gains access to the heme crevice.
• Methemoglobin formation may also occur in vivo as the result of exposure to superoxide anions:
• 2HbFe²⁺O₂ + 2O^2- + 4H⁺---------->2HbFe³⁺ + 3O₂ + 2H₂O
• The formation of methemoglobin may also result from a direct reaction of reduced hemoglobin with the reduction product of the superoxide ion, peroxide:
• 2HbFe²⁺ + 2H₂O₂---------->2HbFe3^+. H₂O + O₂
• As a result of these processes, methemoglobin is formed in normal cells at the rate of about 0.5 to 3% per day.
• Methemoglobin is unable to bind oxygen. It has distinctive pH-dependent spectrum, and in concentrations greater than 10% of the total hemoglobin, imparts to blood a distinctive brownish color that does not disappear on vigorous shaking in air.
• When methemoglobin is present in vivo in concentrations greater than 1.5 to 2.0 g/dl, patients appear visibly cyanotic.
• Methemoglobin combines readily with cyanide to form cyanomethemoglobin, a pigment so stable that it is used in laboratory procedures for quantifying hemoglobin.
• The Drabkin's solution, which is used in hemoglobin assay, contains potassium ferricyanide and potassium cyanide. Potassium ferricyanide changes  Hb to methhemoglobin and potassium cyanide changes methemoglobin to cyanomethamoglobin, which is very stable to be used in photometry.

Hemichrome

· As oxidative denaturation continues, methemoglobin is converted to derivatives known as hemichromes. Hemichromes also may form directly from hemoglobin without methemoglobin as an intermediate.

·  The hemichromes are low-spin, ferric compounds with a greenish hue and a characteristic spectrum.

·    They are formed when the sixth coordination position of iron becomes covalently attached to a ligand within the globin molecule, a change that requires alteration of tertiary protein structure. Probably the most common internal ligand is the so-called distal histidine at E7, the compound so formed has been called a "reversible" hemichrome because relatively mild treatment with reducing agents and dialysis under anaerobic conditions converts it to deoxyhemoglobin. It may not be "reversible" in vivo, however, because it cannot be reduced by methemoglobin reductase.

·  In contrast, the "irreversible" hemichromes cannot be converted back to normal hemoglobin again in vivo or in vitro, implying that more severe distortions of tertiary protein structure have occurred.

·         In one of the irreversible hemichromes, the histidine imidazole groups are protonated – i.e., they participate in hydrogen binding.

·     The other irreversible hemichrome is characterized by a mercaptide and nitrogenous linkage at the fifth and sixth positions. Presumably, the mercaptide link is provided by a cysteine residue in the globin chain, perhaps at b93.

·         As these changes occur in the vicinity of the heme group, oxidative changes also occur in other parts of the hemoglobin molecule.

·       Once the cell's supply of glutathione (GSH) is exhausted, the titrable sulfhydryl groups at b93(F9)Cys are oxidized, often forming a mixed disulfide with glutathione. This change is reversible; however, as further alterations in globin conformation occur, normally protected or "buried" sulfhydryl groups at b112(G14)Cys and a104(G11)Cys become exposed and are oxidized, changes that disrupt at a1b2 contacts. These changes facilitate dissociation of polypeptide chains, first into ab dimers and finally into monomers. In some instances, heme may dissociate from globin, particularly in case of certain unstable hemoglobins.

·     The end products of these changes are precipitated hemichromes and precipitated heme-free globin. In intact erythrocytes, these precipitates take the form of coccoid inclusions known as Heinz bodies, which are not visible with ordinary Wright's stain, but can be easily seen after supravital staining with crystal violet or brilliant cresyl blue. Heinz bodies may become attached to the cell membrane and shorten red cell survival.

Sulfhemoglobin

Another nonfunctional hemoglobin derivative occasionally formed during the oxidative denaturation of hemoglobin is sulfhemoglobin. It is a relatively stable pigment and once formed, cannot be converted to hemoglobin in vivo. Instead, it tends to remain within the cell throughout the cell's life.

Sulfhemoglobin is bright green and has a distinctive spectrum characterized by an absorption band at about 618nm. It is a ferrous compound with one sulfur attached to each heme group. The sulfur is probably attached to a β carbon in the prophyrin ring, forming a thiochlorin.

(Source: Wintrobe's Textbook of Hematology; Tietz's Textbook of Clinical Chemistry)