Positively charged peptides can interact with each other, as revealed by solid phase binding assays

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Solid phase assay systems like ELISA and SPR are widely used to study molecular interactions.
• These methods typically involve monitoring binding between soluble and surface-immobilized molecules.
• Understanding the conditions that facilitate biomolecular interactions is crucial for interpreting assay results.

Purpose of the Study:

• To investigate the interaction between positively charged peptides using solid phase assay systems.
• To determine if immobilization is a prerequisite for detecting interactions between positively charged peptides.
• To compare solid-phase binding data with solution-based methods.

Main Methods:

• Enzyme-linked immunosorbent assay (ELISA) was employed to detect peptide binding.
• Surface plasmon resonance (SPR) was used to quantify the binding kinetics and affinity.
• Nuclear magnetic resonance (NMR) and native gel electrophoresis were used to assess interactions in solution.

Main Results:

• ELISA and SPR confirmed interactions between immobilized positively charged peptides, with dissociation constants (K(d)) between 1 and 14 nM.
• No interaction was observed between positively charged and negatively charged peptides or with bovine serum albumin (BSA).
• Interactions between positively charged peptides were not detected in solution using NMR or native gel electrophoresis.

Conclusions:

• Immobilization of at least one binding partner appears necessary for detecting interactions between positively charged peptides.
• Solid phase assays can reveal interactions that are masked in solution, potentially due to conformational changes upon immobilization.
• These findings highlight the importance of considering immobilization effects when using solid phase assays for studying biomolecular interactions.