Polymeric brushes as functional templates for immobilizing ribonuclease a: study of binding kinetics and activity.

Polymeric brushes as functional templates for immobilizing ribonuclease a: study of binding kinetics and activity. Research Abstract Details 

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Polymeric brushes as functional templates for immobilizing ribonuclease a: study of binding kinetics and activity. Abstract Text:

Sean P Cullen,Xiaosong Liu,Ian C Mandel,Franz J Himpsel,Padma Gopalan,Sean P Cullen,Xiaosong Liu,Ian C Mandel,Franz J Himpsel,Padma Gopalan,

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 mug/cm2 of RNase A was bound to PAA brushes by metal-ion complexation compared to 5.8 mug/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 x 10-14 cm2/s for metal-ion complexation and 0.71 x 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.

Polymeric brushes as functional templates for immobilizing ribonuclease a: study of binding kinetics and activity. Publishing Authors By Initials

SP Cullen,X Liu,IC Mandel,FJ Himpsel,P Gopalan,SP Cullen,X Liu,IC Mandel,FJ Himpsel,P Gopalan,

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Polymeric brushes as functional templates for immobilizing ribonuclease a: study of binding kinetics and activity. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Langmuir : the ACS journal of surfaces and colloid

VOLUME: 24

Page Numbers: 913-20

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ISSN: 0743-7463

DAY: 13

MONTH: 12

YEAR: 2007

Polymeric brushes as functional templates for immobilizing ribonuclease a: study of binding kinetics and activity. Information

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LANGUAGE: eng

NlmUniqueID: 9882736

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Grant and Affiliation Information for Polymeric brushes as functional templates for immobilizing ribonuclease a: study of binding kinetics and activity.

AFFILIATION: Chemical and Biological Engineering, Department of Physics, and Materials Science and Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706.

Country: United States

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MEDLINETA: Langmuir

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