Polymeric Brushes as Functional Templates for Immobilizing Ribonuclease A:  Study of Binding Kinetics and Activity

Abstract

The ability to immobilize proteins with high binding capacities on surfaces while maintaining their activity is critical for protein microarrays and other biotechnological applications. We employed poly(acrylic acid) (PAA) brushes as templates to immobilize ribonuclease A (RNase A), which is commonly used to remove RNA from plasmid DNA preparations. The brushes are grown by surface-anchored atom-transfer radical polymerization (ATRP) initiators. RNase A was immobilized by both covalent esterification and a high binding capacity metal-ion complexation method to PAA brushes. The polymer brushes immobilized 30 times more enzyme compared to self-assembled monolayers. As the thickness of the brush increases, the surface density of the RNase A increases monotonically. The immobilization was investigated by ellipsometry, X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), and near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The activity of the immobilized RNase A was determined using UV absorbance. As much as 11.0 microg/cm(2) of RNase A was bound to PAA brushes by metal-ion complexation compared to 5.8 microg/cm(2) by covalent immobilization which is 30 and 16 times the estimated mass bound in a monolayer. The calculated diffusion coefficient D was 0.63 x 10(-14) cm(2)/s for metal-ion complexation and 0.71 x 10(-14) cm(2)/s for covalent immobilization. Similar values of D indicate that the binding kinetics is similar, but the thermodynamic equilibrium coverage varies with the binding chemistry. Immobilization kinetics and thermodynamics were characterized by ellipsometry for both methods. A maximum relative activity of 0.70-0.80 was reached between five and nine monolayers of the immobilized enzyme. However, the relative activity for covalent immobilization was greater than that of metal-ion complexation. Covalent esterification resulted in similar temperature dependence as free enzyme, whereas metal-ion complexation showed no temperature dependence indicating a significant change in conformation.

Poly(vinylbenzylchloride) beads grafted with polymer brushes carrying hydrazine ligand for reversible enzyme immobilization

“…For this reason, reversible enzyme immobilization via adsorption requires a strong hydrophobic or ionic interaction between the enzyme and support. [4][5][6] Desorption of enzymes from fibrous polymer containing ion-exchange groups was found to require the use of denaturing conditions (under low pH and high ionic strength), but this desorption would be necessary after inactivation of the enzyme upon use. [5][6][7] For reversible enzyme immobilization, the modification of support surface is mostly required to change the character of the base support surface from hydrophobic to hydrophilic in order to create selective absorptive surface for adsorption of protein.…”

“…[4][5][6] Desorption of enzymes from fibrous polymer containing ion-exchange groups was found to require the use of denaturing conditions (under low pH and high ionic strength), but this desorption would be necessary after inactivation of the enzyme upon use. [5][6][7] For reversible enzyme immobilization, the modification of support surface is mostly required to change the character of the base support surface from hydrophobic to hydrophilic in order to create selective absorptive surface for adsorption of protein. [8][9][10] Among the surface functionalization techniques, polymer brushes from surface-initiated polymerizations have been widely used to tailor the surface properties of substrates.…”

Bio-Decorated Polymer Membranes: A New Approach in Diagnostics and Therapeutics

“…There are several examples of polymeric membranes on solid surfaces in which polymer films have been used instead of block copolymers: poly(ortho-phenylenediamine) (PoPD) [19], poly(acrylic acid) [20], poly(N-methyl pyrrole) [21], poly 3,4-ethylenedioxythiophene [22]. The ways in which the immobilization was performed was based on entrapping the biomolecule in the polymer membrane, adsorption by hydrophobic or ionic interactions, or covalent binding to the polymer membrane.…”

“…This system was developed for the removal of RNA in DNA experiments [20]. RNase A, as a one-unit enzyme, cleaves single-stranded RNA.…”

Synthesis and Characterization of Poly(acrylic acid) Brushes: “Grafting‐Onto” Route

“…[1][2][3][4][5][6] Among different types of possible polymers, poly(acrylic acid) brushes are interesting in their own right, with biological applications in biosensors, cell adhesion and protein adsorption. [7][8][9] As a result, their specifi c synthesis has attracted increasing attention during the last decade.…”