Web Release Date: December 13,
Polymeric Brushes as Functional Templates for Immobilizing Ribonuclease A: Study of Binding Kinetics and Activity
and
Chemical and Biological Engineering, Department of Physics, and Materials Science and Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706
Received August 13, 2007
In Final Form: October 17, 2007
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 
g/cm2 of RNase A was bound to PAA brushes by metal-ion
complexation compared to 5.8 g/cm2 by covalent immobilization which is 30 and 16 times the estimated mass bound
in a monolayer. The calculated diffusion coefficient D was 0.63 × 10-14 cm2/s for metal-ion complexation and 0.71
× 10-14 cm2/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.
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