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Processing and immobilization of enzyme Ribonuclease A through laser irradiation

Published online by Cambridge University Press:  17 January 2011

C. Popescu ,
J. Roqueta ,
A. Pérez del Pino ,
M. Moussaoui ,
M. V. Nogués  and
E. György
C. Popescu
Affiliation:
National Institute for Lasers, Plasma and Radiations Physics, 77125 Bucharest, Romania
J. Roqueta
Affiliation:
Consejo Superior de Investigaciones Científicas, Centre d’Investigacions en Nanociència i Nanotecnologia (CSIC-CIN2), Campus UAB, 08193 Bellaterra, Spain
A. Pérez del Pino
Affiliation:
Consejo Superior de Investigaciones Científicas, Institut de Ciència de Materials de Barcelona (CSIC-ICMAB), Campus UAB, 08193 Bellaterra, Spain
M. Moussaoui
Affiliation:
Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
M. V. Nogués
Affiliation:
Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain
E. György*
Affiliation:
National Institute for Lasers, Plasma and Radiations Physics, 77125 Bucharest, Romania; and Consejo Superior de Investigaciones Científicas, Centre d’Investigacions en Nanociència i Nanotecnologia (CSIC-CIN2), Campus UAB, 08193 Bellaterra, Spain
*
a)Address all correspondence to this author. e-mail: eniko.gyorgy@cin2.es
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Abstract

We report the processing and immobilization of enzyme Ribonuclease A (RNase A) onto SiO2 glass collectors using the matrix assisted pulsed laser evaporation (MAPLE) technique. The experiments were performed inside a stainless steel irradiation chamber. A pulsed UV KrF* (λ = 248 nm, τFWHM ≈ 25 ns, ν = 10 Hz) excimer laser source was used for the irradiations. The laser fluence was varied in the range 0.4–0.7 J/cm2. The morphology of the obtained films was investigated by atomic force microscopy (AFM) and their structure and composition by Fourier transform infrared spectroscopy (FTIR). The FTIR spectra of the films obtained from composite MAPLE targets consisting of 1% (w/v) RNase A in Hepes-KOH 10 mM pH 7.5 buffer exhibit the same bands as the spectrum of the initial, nonirradiated material. The enzymatic activity of the obtained structures was analyzed using synthetic substrate polycytidylic acid (poly(C)). The poly (C) cleavage by the immobilized enzyme and the products of formation were analyzed by means of reverse phase high performance liquid chromatography (HPLC).

Keywords

LaserBiomaterialRadiation Effects
Type
Articles
Information
Journal of Materials Research , Volume 26 , Issue 6 , 28 March 2011 , pp. 815 - 821
Copyright
Copyright © Materials Research Society 2011

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