Quantification of the CBD-FITC conjugates surface coating on cellulose fibres
© Pinto et al. 2008
Received: 23 January 2007
Accepted: 09 January 2008
Published: 09 January 2008
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© Pinto et al. 2008
Received: 23 January 2007
Accepted: 09 January 2008
Published: 09 January 2008
Cellulose Binding Domains (CBD) were conjugated with fluorescein isothiocyanate (FITC). The surface concentration of the Binding Domains adsorbed on cellulose fibres was determined by fluorescence image analysis.
For a CBD-FITC concentration of 60 mg/L, a coating fraction of 78% and 110% was estimated for Portucel and Whatman fibres, respectively. For a saturating CBD concentration, using Whatman CF11 fibres, a surface concentration of 25.2 × 10-13 mol/mm2 was estimated, the equivalent to 4 protein monolayers. This result does not imply the existence of several adsorbed protein layers.
It was verified that CBDs were able to penetrate the fibres, according to confocal microscopy and TEM-immunolabelling analysis. The surface concentration of adsorbed CBDs was greater on amorphous fibres (phosphoric acid swollen) than on more crystalline ones (Whatman CF11 and Sigmacell 20).
Due to its high sensitivity and specificity, fluorescence analysis has been extensively used in microarray technology , gene expression monitoring , protein diffusion  or in vivo chemical elements uptake and localization studies . Another area to benefit from fluorescence based techniques is protein quantification. The use of either non-covalent  or covalent labelling [6, 7] increased the detection sensitivity to as low as 40 ng/mL.
Cellulose-Binding Domains (CBD) are modules present in most celulases, being responsible for their high affinity to cellulose crystalline surfaces [8, 9]. The CBD used in this work, produced by limited proteolysis, belongs to cellobiohydrolase I (CBHI) of Trichoderma reesei, as shown in a previous work . Three tyrosine residues define a flat surface, which may be responsible for the affinity to cellulose . This protein has a single amine, the N-terminal of the linker region, which allows a specific reaction with fluorescein isothiocyanate (FITC). The conjugation with FITC does not affect the CBD interaction with cellulose, since the N-terminal is isolated from the cellulose interacting part of the protein. Indeed, the conjugation of FITC does not modify the CBD adsorption isotherms [12, 13]. Since there is only one amine group present in the CBD, the stoichiometry of the conjugation reaction is 1:1. The FITC fluorophore has been attached to antibodies [14, 15], to microparticles  or to other binding domains [12, 17]. Several recombinant CBDs, fused to different proteins, have been produced, as recently reviewed by Shoseyov et al..
Cellulose-Binding Domains (CBD) have been used to target functional molecules to cellulose-containing materials , to improve pulp properties  or as an additive for paper recycling . Bearing in mind that these applications are related to surface effects, in this work we attempted to quantify the CBD surface coverage of cellulose fibres, using the approach based on the use of CBD-FITC previously developed. Our aim was to quantify the protein adsorbed on cellulose fibres and, more specifically, the surface concentration of CBD. This value could, alternatively, be estimated by measuring the specific surface area, by means of the BET isotherm . However, the BET approach is not ideal for porous materials . The presence of CBDs in the interior of the fibres was also investigated.
Amorphous cellulose was prepared, by treating Whatman CF11 with phosphoric acid, which induces the swelling of fibres. As it can be seen in Figure 4, the fibres are larger than the original ones (amorphous Whatman versus Whatman CF11). This swelling effect is due to the disruption or loosening of the microfibrils, thus increasing the volume occupied by the total fibre. As a result, the total surface area available for CBD adsorption increases. Consequently, it is expected that the measured CBD fluorescence would be higher than the one obtained for CF11 fibres, mostly due to an easier penetration of CBD into the fibres structure. This was confirmed, as can be seen in Figure 3. The surface coverage increases about 50% (4 versus 6 layers, respectively for Whatman CF11 and amorphous cellulose). This result can arise either from increased CBD affinity for the more amorphous fibres, or to easier penetration, and hence higher concentration in the fibres. Due to the higher fluorescence obtained with these fibres, the majority of the image exceeded the maximum concentration used in the calibration and it had to be excluded in the image analysis (Fig. 4).
Sigmacell 20 is obtained by the separation of crushed cellulose fibres, with an average size of 20 μm. This mechanical treatment expectedly leads to broken ends or loosen fibrils (amorphous material). Then, the adsorption of CBD is expected to be higher than with CF11 and comparable to the amorphous cellulose (see Figure 3). Again, the fibres present a high fluorescence emission corresponding to a high amount of adsorbed CBD: about 5 layers.
In this work, the surface concentration of CBD adsorbed on cellulose fibres was estimated. The coating values obtained were higher than expected, corresponding in theory to several layers (4 to 6) of CBDs adsorbed on the external surface. However, it has been demonstrated that the CBDs penetrate the fibres. An important amount of CBDs was detected inside the fibres by immunolabelling and confocal microscopy. It seems that a large fraction of the adsorbed CBDs actually penetrate the fibres. The surface coverage values are, undoubtedly, high enough to justify a change in the fibres' surface properties. CBDs may therefore be used as powerful tools to modulate the fibres' surface properties.
Fluorescein isothiocyanate (FITC), SigmaCell Type 20 and Whatman CF11 were obtained from Sigma. Secondary fibres where kindly supplied by Portucel Viana. All chemicals were of the highest purity available in the market.
Amorphous cellulose was prepared by treating Whatman CF11 fibres with phosphoric acid. Briefly, 0.17 g of Whatman CF11 were slowly mixed with 10 mL of cold (4°C) phosphoric acid (85%), and left in contact for 5 minutes. Then, 600 mL of cold water were added and the suspension was filtered through a test sieve (mesh width 71 μm, according to DIN 4188). Finally, the fibres were extensively washed first in tap water and afterwards with distilled water. The obtained material was lyophilized and stored.
The CBDs were prepared according to the following methodology: the Celluclast® commercial enzymatic preparation (Novozymes A/S, Denmark) was digested with Papain (1:1200, protein basis). The CBDs were separated by ultrafiltration through a 10 kDa membrane (Pellicon 2 TFF System from Millipore, USA) and concentrated by precipitation with ammonium sulphate (Merck, Darmstadt, Germany). After dialysis, the protein was injected on a Sepharose Fast-Flow gel (Amersham Pharmacia Biotech AB, Sweden), and the non-adsorbed protein was collected and lyophilized. The purity and identity (CBD from T. reesei CBH I) of this protein has been demonstrated by N-terminal sequencing and MALDI-TOF .
The conjugation of CBD with the labelling probe was carried out by mixing 20 μg of FITC per mg of CBD (2 mg protein/mL in 0.1 M HEPES buffer, pH 9.0). This solution was incubated overnight in the dark, at room temperature, with magnetic stirring. To eliminate the unbound FITC, the labelled CBD mixture was filtered through a BIO-GEL P-4 (BIO-RAD, Hercules, USA) column, previously equilibrated with 50 mM sodium acetate buffer (Panreac, Barcelona, Spain).
Adsorption assays of FITC-labelled CBD were carried out at 4°C. The conjugates were allowed to adsorb on cellulose fibres (20 g/L, in 50 mM sodium acetate buffer, pH 5.0), with continuous magnetic stirring, in the dark, for 2 hours. The supernatant with unbound CBD was removed by centrifugation at 3219 RCF for 10 minutes (Heraeus Megafuge 1.0R). The fibres were washed with acetate buffer to remove the non-adsorbed CBD-FITC.
Fluorescence microscopy observations were performed in a Zeiss Axioskop microscope (Zeiss, Oberkochen, Germany) equipped with a Zeiss AxioCam HRc attached camera (Zeiss, Oberkochen, Germany) and using the AxioVision 3.1 software (Zeiss, Oberkochen, Germany). All images were acquired at 1300 × 1030 pixels and 24 bits colour depths (8 bits per channel). The FITC-CBD quantification was performed as described elsewhere .
Confocal observation was performed in an Olympus (Tokyo, Japan) Fluoview 1000 in Laser Scanning mode equipped with a 60× UPLSAPO lens, with a numerical aperture of 1.35 and a pinhole size of 105 μm.
CBD-specific antibodies were produced in a rabbit (Oryctolagus cuniculus) maintained under standard conditions of housing with unrestricted access to food and water; these conditions followed European Union Directive no. 86/609/CEE. Briefly, the rabbit was immunized intradermically (i.d.) with a 1:1 suspension of Phosphate Buffered saline (PBS)/Complete Freund's adjuvant containing 500 μg CBD and boosted two weeks later i.d. with a 1:1 suspension of PBS/Incomplete Freund's adjuvant containing 500 μg CBD. Blood was collected three weeks after the second immunization for the preparation of immune serum. Purification of IgG antibodies from this serum sample was performed as follows: the serum sample was equilibrated in a binding buffer (20 mM sodium phosphate, pH 7.0) by overnight dialysis and 3 ml of this preparation was applied to a Protein G HP affinity column (HiTrap, Amersham Biosciences, UK). Bound antibodies were eluted with Glycine -HCl buffer, pH 2.7 and recovered in 50 μl of 1 M Tris-HCl pH 9.0 per ml of eluent, according to the manufacturer's instructions. Recovered IgG antibodies were further equilibrated in PBS in a VIVAPORE concentrator with a 7.5 kDa cutoff membrane (Vivascience, Hanover, Germany) and stored at -80°C in frozen aliquots. The anti-CBD antibody titre of this preparation was determined by ELISA. Specific anti-Sap2 or anti CaS antibodies in mice sera collected by retrorbital bleeding were quantified by ELISA. Polystyrene microtitre plates (Nunc, Roskilde, Denmark) were coated with 20 μg/ml of CBD and incubated o.n. at 4°C. Wells were then saturated for 1 h at room temperature with 1% BSA in PBS. Serial dilutions of the serum samples were then plated and incubated for 2 h at room temperature. After washing, bound antibodies were detected by adding alkaline phosphatase-coupled monoclonal goat anti-rabbit-IgG antibody (Southern Biotechnology Associates, Birmingham, ALA, USA) for 30 min at room temperature. Substrate solution containing p-nitrophenyl phosphate (Sigma, St. Louis, USA) was then added after washing and the reaction was stopped by adding 0.1 M EDTA pH 8.0. The absorbance was measured at 405 nm. The ELISA antibody titres were expressed as the reciprocal of the highest dilution giving an absorbance of 0.1 above that of the control (no serum added). The titre of anti CBD antibodies in the purified IgG preparation was of 4014. No antibodies with this specificity were detected in the control sera from non-immunized rabbits.
The CBD-treated Whatman CF11 fibres were fixed in a freshly prepared mixture of 0.2% glutaraldehyde (v/v), 2% paraformaldehyde (w/v) in 0.05 M phosphate buffer (pH 7–7.2). Successive periods of vacuum (5 to 10 min) and air inlet were carried out, up to two hours. Afterwards, the fibres were washed 3 × 10 minutes with 0.05 M phosphate buffer. The samples were then dehydrated through a graded series of ethanol and embedded in London Resin White (hard mixture) polymerized for 24 h at 50°C.
Immunolabelling was done on ultrathin transverse sections (500 Å) floating on plastic rings . The sections were floated on several dilutions of the antiserum in 10 mM Tris buffered saline (500 mM NaCl) to determine the optimal ratio of labelling and background . The secondary marker was Protein A-gold (pA G5, BioCell). The gold particles were further enhanced using a silver enhancing Amersham kit. Finally, the thin-sections were transferred on copper-grids, post-stained with 2.5% aqueous uranyl acetate and examined with a Philips CM 200 Cryo-TEM at an accelerating voltage of 80 kV.
To guarantee semi-quantitative comparative labelling, experiments were carried out in parallel on treated and non-treated samples of CBD. Therefore, the exposure to the antibody was identical.
Ricardo Pinto was supported by a grant from the Fundação para a Ciência e a Tecnologia (FCT) SFRH/BD/6934/2001.
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