Passive Absorption — Looking at Effects of Charge

There are two major groups of chemicals that are passively absorbed across biological membranes: 1) lipophilic, uncharged molecules and 2) lipophilic, charged molecules.

Lipophilic, uncharged (neutral) herbicide molecules such as monuron, norflurazon, oryzalin, and triazines, can passively diffuse into plant cells. The lipophilic nature of these herbicides allows them to diffuse rapidly across the lipid bilayer of plant membranes as driven by the concentration gradient between the external herbicide solution and the cell interior. Net movement stops when concentrations reach equilibrium between the external solution and the cell interior.

We know that uncharged, lipophilic herbicides cross membranes via passive diffusion based on experimental evidence which has shown:

• herbicide absorption saturates over time to reach equilibrium concentrations inside and outside the cell,
• herbicide absorption rates increase linearly with external herbicide concentrations,
• herbicides rapidly efflux out of the cell toward an external solution with lower concentration,
• temperature coefficients (Q₁₀) of absorption are less than 2 which indicates energy is not required, and
• herbicide absorption is insensitive to metabolic inhibitors because energy is not required for passive transport. 

Lipophilic, charged (ionic) molecules such as bentazon, 2,4-D, clopyralid, chlorsulfuron, imazapyr, and sethoxydim, are either weak acids or weak bases. In an aqueous solution, neutral weak acids (R-COOH, where R is the remainder of the molecule; also referred to as HA) are in equilibrium with their charged conjugate base (R-COO^-; also referred to as an anion, A^-)  (equation 1). Neutral weak bases (R-NH₂) are in equilibrium with their charged conjugate acid (R-NH₃⁺)  (equation 2). The relative concentrations of the hydrophobic, undissociated acid molecules (HA, also referred to as R-COOH) or the more polar anions (A^-, also referred to as R-COO^-) depend on the solution pH and the strength of the acid as described by the Henderson-Hasselbach equation (equation 3). The ability of these molecules to be either neutral or charged can greatly increase their rates of movement across plant membranes as well as their accumulation inside the cell.

Equations for the distribution of weak acids and bases in solution and its dependence on pH:

1. Neutral weak acid and its conjugate base: R-COOH  ↔  R-COO^-  +  H⁺
2. Neutral weak base and its conjugate acid:  R-NH₂  +  H₂O ↔ R-NH₃⁺  +  OH^-
3. Henderson-Hasselbach equation:  pH  =  pKa  +  log  [A^- ]/[HA]

Thinking Question:

Which of these herbicides will be more lipophilic at pH 7? At pH 2? How will this influence their membrane permeability?

• Atrazine (pKa = 1.7)
• Bentazon (pKa = 3.58)
• Monuron (pKa = none)

• ANSWER: At pH 7, atrazine will be more lipophilic; as a weak base with a pKa of 1.7, its amine group (-NH₂) is charged only at very acidic pHs (-NH₃⁺). Atrazine is very lipophilic under most physiological pHs, traversing membranes readily.
• At pH 2, more of the bentazon molecules (pKa 2.45) will be in their acid, protonated form, and thus, will be more lipophilic. Proportionately, more bentazon molecules will be able to traverse lipophilic membranes when in solutions or cell compartments of lower pH, as compared to more alkaline pHs where more of the bentazon molecules will be anions, and more hydrophilic, less likely to traverse cell membranes.
• Monuron will be lipophilic regardless of pH because it lacks functional groups affected by pH, and is thus non-ionizable.