Hemoglobin (Hb) is the chief protein of the red blood cells. A single red blood cell contains about 280 million molecules of hemoglobin (Hb). Hb can give rise to many derivatives which are discussed below.
Hemoglobin exists in multiple structural and functional forms, and its derivatives are broadly classified into:

1. Functional derivatives: HbO₂, HbCO₂, HHb (oxygenated, carbamino, and deoxygenated forms of Hb involved in normal physiology)
2. Dysfunctional derivatives: COHb, MetHb, SulfHb, HbNO, HbA1c (abnormal forms that impair oxygen transport or alter Hb function)

   1. Action of Acids

Acids split Hb into globin and heme (containing ferrous iron, Fe²⁺, therefore also called ferroheme). In the presence of O₂, the ferrous iron may be oxidized to ferric iron (Fe³⁺), forming ferriheme, which can bind negatively charged acid anions.

Reaction (Sahli’s method principle):

• Hb + 2HCl → Globin + Hematin (acid hematin)
• (Fe²⁺ → Fe³⁺ oxidation may occur depending on conditions)

Key points:

1. Acid hematin is a brown-colored compound (responsible for color change in acid hemoglobin estimation).
2. This reaction is the basis of Sahli’s method for estimation of hemoglobin (a simple colorimetric method used in basic labs).
3. Acid hematin formation is an irreversible denaturation process under strong acidic conditions (protein structure is destroyed).
4. Hematin formed is also referred to as acid hematin chloride complex in older literature (represents Hb + acid combination product).

   2. Action of Alkalies

Alkalies also split Hb into globin and ferroheme. The heme iron is oxidized to ferric form (Fe³⁺), which combines with hydroxyl groups to form alkaline hematin (hematin hydroxide complex).

Reaction:

• Hb + NaOH → Globin + Alkaline hematin (hematin-OH complex)

Key points:

1. This compound is brownish in color (similar pigment formation as acid hematin).
2. Alkaline hematin formation represents irreversible denaturation of hemoglobin under alkaline conditions (loss of oxygen-binding structure).
3. Alkaline hematin is also referred to as hematin hydroxide complex or alkaline hematin base (different nomenclature in literature).

   3. Reaction with CO₂

This leads to the formation of carbamino compounds.

Reaction:

• Hb-NH₂ + CO₂ ⇌ Hb-NH-COO⁻ + H⁺
  (formation of carbaminohemoglobin, HbCO₂)

Key points:

1. CO₂ binds to terminal amino groups of globin, not to heme (globin chains participate in binding).
2. This is a reversible reaction important for CO₂ transport (about 10–20% of CO₂ is carried this way) (rest mainly as bicarbonate).
3. This reaction contributes to the Haldane effect, where deoxygenated hemoglobin binds more CO₂ (important in tissue gas exchange).
4. It also contributes indirectly to acid–base balance via H⁺ formation (helps buffer blood pH).
5. Majority of CO₂ transport occurs as bicarbonate (HCO₃⁻) in plasma (via carbonic anhydrase in RBCs).

   4. Reaction with CO

This results in the formation of carboxyhemoglobin (HbCO or CO-Hb).

Reaction:

• Hb(Fe²⁺) + CO ⇌ HbCO

Key points:

1. Endogenous CO is produced during heme degradation by heme oxygenase:
   • Heme + O₂ + NADPH → Biliverdin + Fe²⁺ + CO
   (biliverdin is converted to bilirubin by biliverdin reductase) (this pathway is continuous in normal RBC turnover)
2. In heavy smokers, up to 5–10% COHb may be present; in non-smokers 1–2% is detectable (baseline exposure varies with environment).
3. Affinity of Hb for CO is ~200–250 times higher than for O₂ (explains strong toxicity of CO).
4. COHb is highly stable and dissociates slowly (leading to prolonged hypoxia).

Toxic effects of COHb:

1. COHb cannot carry oxygen → decreased O₂ carrying capacity of blood (functional anemia state).
2. Left shift of O₂-Hb dissociation curve → impaired oxygen release to tissues (tissues receive less oxygen despite normal levels).
3. Allosteric stabilization of R-state hemoglobin → further reduces O₂ unloading (prevents oxygen release in tissues).
4. CO inhibits cytochrome c oxidase (Complex IV) → histotoxic hypoxia (blocks cellular respiration at mitochondrial level).
5. CO exposure can also cause delayed neurological injury due to oxidative stress and lipid peroxidation after reperfusion (seen after recovery phase in severe poisoning).

Clinical features:

• Cherry-red coloration of blood (classic but not always clinically visible) (more evident post-mortem than in living patients)
• Headache, dizziness, confusion, loss of consciousness (progresses with exposure level)

Treatment:

• 100% oxygen or hyperbaric oxygen therapy (to rapidly displace CO from Hb)
• Reaction: HbCO + O₂ → HbO₂ + CO
• Hyperbaric oxygen increases dissolved plasma oxygen independent of Hb (supports tissue oxygenation even when Hb is saturated with CO)

   5. Reaction with Oxidizing Reagents

Oxidizing agents such as ferricyanide [Fe(CN)₆]³⁻, nitrites (NO₂⁻), chlorates, peroxides convert hemoglobin to methemoglobin (MetHb).

Reaction:

• Hb(Fe²⁺) → MetHb (Fe³⁺)

Key points:

1. Ferric iron (Fe³⁺) cannot bind oxygen (loss of oxygen-carrying ability).
2. Methemoglobin is dark brown in color (characteristic chocolate-brown appearance).
3. MetHb is physiologically present in small amounts (normally kept low by enzyme systems).

Reduction back to Hb:

• MetHb + NADH + H⁺ → Hb + NAD⁺
  (via cytochrome b5 reductase system – major pathway) (primary protective mechanism in RBCs)

Additional pathways:

1. Glutathione-dependent reduction system (secondary antioxidant defense)
2. NADPH-dependent methemoglobin reductase system (enhanced by methylene blue) (drug-assisted pathway)

Normal physiology

1. ~1–2% of Hb is continuously oxidized to MetHb (constant but controlled oxidation)
2. Major reducing system:
   • NADH-dependent cytochrome b5 reductase (most important enzyme in RBCs)
3. Minor system: NADPH-dependent pathway (becomes important in therapy)
4. Normal MetHb level: <1–2% (≈1%) (kept stable under healthy conditions)

Methemoglobinemia

Causes:

1. Congenital deficiency of cytochrome b5 reductase (inherited enzymatic defect)
2. Hemoglobin M variants (HbM disease) (abnormal globin stabilizes Fe³⁺ state)
3. Oxidizing agents: nitrites, nitrates (well water), sulfonamides, aniline dyes, dapsone, benzocaine, etc. (drug-induced common cause)

Effects:

1. Reduced oxygen delivery (functional anemia) (Hb present but non-functional)
2. Left shift of O₂-Hb dissociation curve (impairs oxygen release to tissues)
3. Normal PaO₂ but tissue hypoxia (important clinical feature) (key diagnostic clue)

Clinical features:

• Cyanosis unresponsive to oxygen (classic sign)
• Chocolate-brown blood (diagnostic appearance)
•  Dyspnea, headache, dizziness (due to tissue hypoxia)

Treatment

1. Methylene blue (1–2 mg/kg IV)

• Acts via NADPH-dependent reduction system (accelerates conversion of MetHb to Hb)
• Reaction:
• Methylene blue (oxidized) → Leucomethylene blue (reduced) → reduces MetHb to Hb

2. Ascorbic acid (adjunct antioxidant) (slow but supportive reduction of MetHb)

Important caution:

• In G6PD deficiency: methylene blue may be ineffective or cause hemolysis due to reduced NADPH availability (risk of oxidative RBC damage)

Cyanide binding

MetHb binds cyanide to form cyanomethemoglobin:

• MetHb + CN⁻ → CyanometHb

Nitrite therapy basis:

1. Nitrites induce MetHb formation (therapeutic induction of MetHb)
2. MetHb binds cyanide and detoxifies it (protects cytochrome oxidase system)

Final detoxification:

• CN⁻ + S₂O₃²⁻ → SCN⁻ (thiocyanate)

(via rhodanese enzyme in mitochondria) (thiocyanate is less toxic and excreted in urine)

Key mechanism:

• Cyanide inhibits cytochrome c oxidase (Complex IV), causing cellular hypoxia despite normal oxygen content in blood (cellular respiration failure)

   6. Formation of Sulfhemoglobin

It can be formed by incorporation of sulfur into Hb in presence of H₂S or sulfur-containing drugs.

Reaction:

• Hb + sulfur compounds (H₂S / drugs) → Sulfhemoglobin (SulfHb) (irreversible modified Hb pigment)