Download Effect of sol-gel encapsulation on the spectroscopic and

Effect of sol-gel encapsulation on the spectroscopic and electrochemical properties
of horse cyt c.
Deriu D., Pagnotta S.E., Santucci R. and Rosato N.
Departement of Experimental Medicine and Biochemical Sciences
University of Rome, "Tor Vergata", Via Montpellier 1, 00133 Italy
Encapsulation of biological molecules, as enzymes or other proteins, into inorganic host materials
using sol-gel processes has attracted the attention of several researchers over the past few years. These
new composite materials incorporate the chemically selective functionality and reactivity of different
biomolecules in solid state form and have led to the perspective of developing a new class of solid state
chemical and biomedical sensors (1). The use of sol-gel derived silicate materials for protein
immobilisation proteins offers three major advantages:1) samples achieved are transparent, thus
suitable for the application of chemical and biochemical sensors that rely on changes of the absorbance
and/or fluorescence signal, 2) the material can be chemically modified with various polymer additives,
redox modifiers and organically modified silanes, leading to an electrically conducting material with
high potentialities for the constructing of electrochemical sensors,3) sol-gels have tuneable pore size
and pore distribution, which allows small molecules and ions to diffuse into the matrix, whereas large
biomolecules remain trapped in the pores. A number of review articles have been published in the solgel- based biosensing field (2,3), and our attention was focussed on electrochemical transduction based
on biosensors. For example, an enzyme capable of direct electron transfert even when immobilized on
an electrode surface, will permit electrochemical measurement of the enzyme substrate without
addition of a mediator on the electrode surface or into the analyte solution.
Horse heart cytochrome c is one of the most studied heme-containing redox proteins; its properties
have been investigated under a variety of experimental conditions, and its behaviour in solution is well
known. In the present work, we have immobilized horse cytochrome c (cyt c) on the surface of a
graphite pyrolytic (GP) electrode using a calcium-modified silica matrix synthesized with the sol-gel
technique. As illustrated in fig.1, the protein immobilized on a GP electrode at pH 7.0, shows quasireversible voltammetric behaviour in the Fe(III)/Fe(II) redox process. To investigate the system
stability, cyclic Voltammogram(CV) of the samples was run periodically for one week; notably, the
immobilized protein retained its redox activity.
To determine the effect of calcium nitrate-doped-sol-gel on cyt c conformation and to establish if the
protein retains its native structure, UV-VIS and circular dichroism (CD) spectroscopies were performed
during gelation time. Since native cyt c shows a typical absorbtion spectrum,any environmental change
inducing small structural alteration in the protein will appear as wavelenght shifts in the optical
spectrum (4)
According with previous work, soluble and entrapped cyt c (pH range : 5.5- 6.0) shows an identical the
same UV-Vis spectrum(5). Similar results were obtained with CD.
Also, unfolding of the entrapped protein induced by microenvironment changes was defined by dipping
the samples in an acidic 0.04MHCl solution. The denaturation process was followed by measuring the
shift of the Soret maximum, from  = 408 nm (typical of the native protein) to 395 nm,(typical of
unfolded protein).
The refolding process, followed by adding a 50mM Tris- HCl pH 7, to the sample, proved that
unfolding-refolding process of immobilized cyt c is fully reversible, under the conditions investigated
as data of fig 2 (panel a and b) clearly show.
Though still preliminary, data obtained on cyt c encapsulated in calcium nitrate-doped-silica appear to
possess high potentialities in the prospective of a successful application in biosensoristic area.
References
1. W.Jin, J.D. Brennan, Anal. Chim. Acta 461(2002) 1.
2. B.D MacCraith, C.McDonagh, A.K.McEvoy, T.Butler, G.O' Keeffe, V.Murphy,J.Sol-Gel Sci.
Technol.8 (1997)1053.
3. J.Lin, C.W. Brown, Trends Anal. Chem. 16 (1997) 200.
4. J. M. Miller, B.Dunn, J.S. Valentine, J. I. Zink., J. Non-Cryst. Solids, 202 (1996) 279.
5. E. Droghetti, G. Smulevich, J.Biol. In. Chem., 10 (2005) 696.
Fig 1: Cyclic voltammogram of sol-gel-entrapped horse cyt c, immobilized on a GP electrode, run in
0.1 M phosphate buffer, pH 7.0 Scan rate: 1,5 V/s.
410.0
410.0
407.5
407.5
405.0
405.0
 max
 (max)
The cyclic voltammogram, periodically registered for a week, remained practically unchanged.
402.5
402.5
400.0
400.0
397.5
397.5
395.0
0
40
80
120
160
395.0
0
50
150
200
250
300
350
time (hours)
time (min)
a
100
b
Fig 2:
Panel a: Shift of cyt c max in the Soret band as function of time in presence of an acidic 0.04M HCl
solution
Panel b: Shift of cyt c max in the Soret band as function of time during the refolding process
followed by adding a 50mM Tris- HCl pH 7.