• Proteins are among the most abundant (plentiful) buffers in the body.
• They are especially abundant inside the cells, where their concentration is very high.
• The intracellular pH is slightly lower than the extracellular fluid (ECF) pH.
• However, changes in intracellular pH occur approximately in proportion to changes in extracellular fluid pH.
• There is a small diffusion of H⁺ and HCO₃⁻ across the cell membrane.
• H⁺ and HCO₃⁻ require several hours to reach equilibrium with the extracellular fluid.
• Exception: In red blood cells (RBCs), equilibrium occurs rapidly.
• CO₂ diffuses rapidly through all cell membranes.
• The movement of CO₂, H⁺, and HCO₃⁻ (elements of the bicarbonate buffer system) causes the intracellular pH to change whenever extracellular pH changes.
• Therefore, intracellular buffer systems help prevent changes in extracellular fluid pH.
• However, these intracellular buffer systems require several hours to become maximally effective.
• In red blood cells, hemoglobin (Hb) is an important intracellular buffer.
• The buffering reaction is: H⁺ + Hb ⇌ HHb
• Reaction Explanation:
  • When H⁺ increases, hemoglobin (Hb) binds H⁺ to form HHb.
  • When H⁺ decreases, HHb releases H⁺, forming Hb again.
  • This reversible reaction helps minimize changes in pH.
• Approximately 60% to 70% of the total chemical buffering of body fluids occurs inside the cells.
• Mathematical Calculation:
  • Minimum intracellular buffering = 60%
  • Maximum intracellular buffering = 70%
  • Therefore, about 6 to 7 out of every 10 parts of total body fluid buffering occur inside cells.
• Most of this intracellular buffering is produced by intracellular proteins.
• Except for red blood cells, the slow movement of H⁺ and HCO₃⁻ through cell membranes delays the maximum buffering action of intracellular proteins.
• Therefore, maximum buffering of extracellular acid–base disturbances by intracellular proteins may take several hours.
• Another reason proteins are effective buffers is their high concentration inside cells.
• In addition, the pK values of many intracellular proteins are close to the normal intracellular pH.
• Therefore, protein buffer systems work efficiently inside cells.

KEY CONCEPT

• Proteins are the most abundant intracellular buffers because of their high concentration inside cells.
• Intracellular pH changes approximately in proportion to extracellular fluid pH.
• H⁺ and HCO₃⁻ diffuse slowly across cell membranes and require several hours to reach equilibrium, except in red blood cells, where equilibrium is rapid.
• CO₂ diffuses rapidly through all cell membranes.
• Hemoglobin (Hb) in red blood cells is an important intracellular buffer.
• Buffer reaction: H⁺ + Hb ⇌ HHb.
• Approximately 60–70% (6–7 out of every 10 parts) of total body fluid chemical buffering occurs inside cells, mainly due to intracellular proteins.
• Protein buffers are highly effective because their pK values are close to intracellular pH.

Isohydric Principle: All Buffers in a Common Solution Are in Equilibrium With the Same H⁺ Concentration

• The buffer systems in the body do not work separately.
• All buffer systems work together because they all involve the same H⁺ (hydrogen ion).
• Therefore, when the H⁺ concentration changes in the extracellular fluid, all buffer systems change their balance at the same time.
• This is called the Isohydric Principle.
• The Isohydric Principle is represented by the following equation:

$$[H^+] = K_1 \times \frac{HA_1}{A_1^-} = K_2 \times \frac{HA_2}{A_2^-} = K_3 \times \frac{HA_3}{A_3^-}$$[H+]=K1​×A1−​HA1​​=K2​×A2−​HA2​​=K3​×A3−​HA3​​

Solving the Biological Equation into the Easiest Conceptual Understanding

Step 1: Understand Each Symbol

Symbol	Meaning
H⁺	Hydrogen ion concentration (acidity)
K₁, K₂, K₃	Dissociation constants of different acids
HA₁, HA₂, HA₃	Acid form of Buffer 1, Buffer 2, Buffer 3
A₁⁻, A₂⁻, A₃⁻	Base form of Buffer 1, Buffer 2, Buffer 3

Step 2: Expand the Equation

The equation actually means:

Buffer System 1

$$[H^+] = K_1 \times \frac{HA_1}{A_1^-}$$[H+]=K1​×A1−​HA1​​

Meaning:

Hydrogen ion concentration depends on:

• Dissociation constant (K₁)
• Amount of acid (HA₁)
• Amount of base (A₁⁻)

Buffer System 2

$$[H^+] = K_2 \times \frac{HA_2}{A_2^-}$$[H+]=K2​×A2−​HA2​​

Meaning:

The same H⁺ concentration is also maintained by Buffer System 2.

Buffer System 3

$$[H^+] = K_3 \times \frac{HA_3}{A_3^-}$$[H+]=K3​×A3−​HA3​​

Meaning:

The same H⁺ concentration is also maintained by Buffer System 3.

Step 3: What Does the Equal (=) Sign Mean?

The equal signs do not mean that:

• K₁ = K₂ = K₃

or

• HA₁ = HA₂ = HA₃

Instead, they mean:

Every buffer system is maintaining the same H⁺ concentration in the body fluid.

So,$$\boxed{\text{Same } H^+ \text{ is shared by all buffer systems}}$$Same H+ is shared by all buffer systems​

Step 4: Easy Concept

Imagine there are three workers lifting one heavy box.

• Worker 1 = Bicarbonate buffer
• Worker 2 = Phosphate buffer
• Worker 3 = Protein buffer

The box = H⁺ ions.

If the box becomes heavier (more H⁺),

➡️ All three workers immediately help together.

No worker works alone.

Similarly,

When H⁺ changes, all buffer systems adjust together.

This is the Isohydric Principle.

Step 5: What Happens if One Buffer Changes?

Suppose the bicarbonate buffer removes H⁺.

Immediately,

• Protein buffer releases some H⁺.
• Phosphate buffer also adjusts its H⁺.

Now,

All buffers reach a new equilibrium.

Suppose the protein buffer binds more H⁺.

Immediately,

• Bicarbonate buffer changes.
• Phosphate buffer changes.

Again,

All buffers become balanced together.

Step 6: Meaning of “Buffers Buffer One Another”

The buffer systems help each other.

They continuously transfer H⁺ ions between themselves.

Example:

Protein Buffer ←→ H⁺ ←→ Bicarbonate Buffer ←→ H⁺ ←→ Phosphate Buffer

Hydrogen ions move between buffer systems until all are balanced.

Step 7: Final Meaning of the Equation

The equation says:

• Every buffer has its own acid (HA) and base (A⁻).
• Every buffer has its own dissociation constant (K).
• But all buffers share the same H⁺ concentration.
• Therefore, changing one buffer automatically changes all the others.

• K₁, K₂, and K₃ are the dissociation constants of the three different acids:
  • HA₁
  • HA₂
  • HA₃
• A₁⁻, A₂⁻, and A₃⁻ are the base forms (free negative ions) of the three buffer systems.
• Therefore, each buffer system has its own acid, base, and dissociation constant, but all of them are linked by the same H⁺ concentration.
• The implication of the Isohydric Principle is that if the balance of one buffer system changes, the balance of all the other buffer systems also changes.
• This happens because all buffer systems continuously exchange H⁺ ions with one another.
• Therefore, the buffer systems help (buffer) one another by shifting H⁺ ions back and forth until a new equilibrium is reached.

KEY CONCEPT

• All buffer systems work together because they share the same H⁺ concentration.
• This concept is called the Isohydric Principle.
• The equation: $$[H^+] = K_1 \times \frac{HA_1}{A_1^-} = K_2 \times \frac{HA_2}{A_2^-} = K_3 \times \frac{HA_3}{A_3^-}$$[H+]=K1​×A1−​HA1​​=K2​×A2−​HA2​​=K3​×A3−​HA3​​ means that every buffer system maintains the same H⁺ concentration, even though each has its own acid (HA), base (A⁻), and dissociation constant (K).
• Changing one buffer system automatically changes all the others, because they exchange H⁺ ions until equilibrium is restored.