Polymeric brushes as functional templates for immobilizing ribonuclease A: study of binding kinetics and activity.
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.
Cullen
Sean P
SP
Chemical and Biological Engineering, Department of Physics, and Materials Science and Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Liu
Xiaosong
X
Mandel
Ian C
IC
Himpsel
Franz J
FJ
Gopalan
Padma
P
D000180:Acrylic Resins; D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D005898:Glass; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism*; D012825:Silicon; D013057:Spectrum Analysis; D013816:Thermodynamics; D014965:X-Rays
D000180:Acrylic Resins; D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; C006903:carbopol 940; D012259:Ribonuclease, Pancreatic; D012825:Silicon