Hydrophobic agaroses: basis for a model of multivalent effector-receptor interactions

Area of Science:

• Biochemistry
• Biophysical Chemistry

Background:

• Understanding enzyme-ligand interactions is crucial in biochemistry.
• Sepharose 4B is a common chromatography matrix for protein purification.

Purpose of the Study:

• To investigate the effect of butyl residue density on Sepharose 4B on phosphorylase b binding affinity.
• To explore the cooperativity of enzyme adsorption and derive a model for effector-receptor interactions.

Main Methods:

• Affinity chromatography using butyl-Sepharose 4B gels with varying degrees of substitution.
• Enzyme binding studies with phosphorylase b.
• Analysis of binding data using Hill coefficients to determine cooperativity.

Main Results:

• Increased butyl density on Sepharose 4B enhanced phosphorylase b affinity, with a Hill coefficient of 2.9 suggesting positive cooperativity (≥3 binding sites).
• Affinity decreased with fractional saturation, indicating negative cooperativity (Hill coefficient of 0.44) due to multivalent binding.
• A multivalent, mobile receptor model was derived to explain positive and negative cooperativity, high binding constants, and low dissociation rate constants.

Conclusions:

• The derived multivalent, mobile receptor model successfully explains complex effector-receptor interactions.
• The model's applicability was validated using literature data on concanavalin A and cholera toxin binding to fat cells.
• This suggests the postulated interaction mode is biologically relevant for multivalent effectors.