Preparation, morphological characterization, and activity of thin films ofhorseradish peroxidase

Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/ SiO2 or glass supports previously activated by sila nization and succinylation. Labeling by fluorescent or by Electron Spin Res onance (ESR) probes was used to quantify the surface density of active grou ps and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-unifor m protein adsorption due to physical interactions was present when the supp orts were not activated for covalent binding and was, in large part, remove d by washing. The enzyme deposited by covalent binding formed homogeneous l ayers with a height in the range 60-90 Angstrom. By using a fluorescent lab el, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/ SiO2, corresponding to an estimated area per molecule of 2800 Angstrom(2) w hich is in agreement with the value expected on the basis of the crystallog raphic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x 10(12) molecu les cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K-M be ing of the order of 3-5x10(5) M-1 s(-1) that is 1/20th of free HRP. The hal f-life time of the activity of the enzyme immobilized by covalent binding w as longer than 40 days at 6 degrees C. (C) 2000 John Wiley & Sons, Inc.