Polyvinyl alcohol-glutaraldehyde network as a support for protein ...

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Introduction. Immobilisation of biomaterials has been widely used in many fields, such as industry, biochemistry and immunology (Cheetham, 1985; Hermanson ...
Biotechnology Techniques, Vol 11, No 2, February 1997, pp. 67–70

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Polyvinyl alcohol-glutaraldehyde network as a support for protein immobilisation A.M. Araujo1,2, M.T. Neves Jr.1, W.M. Azevedo3, G.G. Oliveira1, D.L. Ferreira Jr.1, R.A.L. Coelho1, E.A.P. Figueiredo1 and L.B. Carvalho Jr1* 1

Laboratório de Imunopatologia Keizo Asami and Departamento de Bioquímica, Universidade Federal de Pernambuco (UFPE), Cidade Universitária, 50670–910, Recife, PE, Brasil; 2HEMOPE – Fundação de Hematologia e Hemoterapia de Pernambuco; 3Departamento de Química Fundamental/UFPE

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Polyvinyl alcohol-glutaraldehyde (PVA-glut) network was synthesised in bead and disc forms and used for protein immobilisation. Xanthine oxidase, a-amylase and amyloglucosidase were covalently fixed on the beads yielding preparations with specific activities retention of 72.3%, 1.6% and 1.4%, respectively. Km of xanthine oxidase PVA-glut beads (24 ± 4 µM) was slightly higher than that estimated for the soluble enzyme (16 ± 2 mM). Antigens from Schistosoma mansoni were covalently fixed onto PVA-glut discs and ELISA was carried out. The relationship between the amount of fixed antigen and the results obtained by ELISA showed a hyperbolic curve and the saturation of antigen-antibody complex was achieved at the plateau.

Introduction Immobilisation of biomaterials has been widely used in many fields, such as industry, biochemistry and immunology (Cheetham, 1985; Hermanson et al., 1992). This technology employs a large number of natural and artificial materials as matrices (Kennedy and Cabral, 1987). In our laboratory, we have proposed immobilisation procedures using dacron (Carneiro Leão et al., 1994), polyaniline (Nadruz et al., 1996) and glyptal (Jordão et al., 1996). Here, polyvinyl alcoholglutaraldehyde (PVA-glutaraldehyde) network is described for antigen and enzyme immobilisation. Previously, this material was used for the diagnosis of plague as a solid-phase in ELISA (Araujo et al., 1996) and also in laser induced fluorescence (Carvalho et al., 1996). Materials and methods Synthesis of PVA-glutaraldehyde beads Polyvinyl alcohol (2.0 g; Reagen®) was dissolved in deionised water (20 mL) containing sodium dodecylsulfate (10 mg; Sigma) under heating and stirring. Glutaraldehyde (25%, w/v; 4.12 mL; Sigma) and H2SO4 (0.3 M; 1 mL) were successively added to the dissolved PVA and then the solution was slowly poured into a beaker containing mineral oil/water mixture (45:45; v/v) with stirring. After about 15 min, PVA-glutaraldehyde beads were synthesised. In order to remove oil and © 1997 Chapman & Hall

sodium dodecylsulfate the beads were exhaustively washed with ethanol 95% v/v (500 mL) and deionised water (1 L). Synthesis of PVA-glutaraldehyde discs According to Araujo et al. (1996). Enzyme immobilisation a-Amylase (20 mL of 65 U/mg protein; Termamyl, Novo Nordisk, Paraná, Brazil) prepared in 50 mM citrate-phosphate buffer, pH 4.5, was incubated with PVA-glutaraldehyde beads (1 g) under stirring overnight at 4°C. After immobilisation, the water insoluble enzymatic derivative was washed with 1 M NaCl (100 mL) in buffer. Amyloglucosidase (20 mL of 6.5 U/mg protein; AMG, Novo Nordisk, Paraná, Brazil), prepared in 50 mM citrate-phosphate buffer, pH 6.5, was incubated with PVA-glutaraldehyde as described above. Xanthine oxidase (10 mL; Nutritional Biochemichals Corporation) prepared in 0.1 M phosphate buffer, pH 8.0, was incubated with PVAglutaraldehyde beads (0.6 g) under mild stirring overnight at 4°C. Then, the immobilised enzymatic derivative was exhaustively washed with deionised water and 1 M NaCl, successively. Afterwards, overnight incubation with 1 M glycine was carried out to inactivate free carbonyl groups and washings were carried out again. Biotechnology Techniques · Vol 11 · No 2 · 1997

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Antigen immobilisation Soluble worms adult proteins (SWAP) of Schistosoma mansoni were obtained according to Mott and Dixon (1982) and were covalently immobilized onto PVAglutaraldehyde discs according to Araujo et al. (1996) at concentrations ranging from 0.039 to 5 mg per disc. Positive sera were collected from individuals 40 days after they were accidentally infected by S. mansoni, whereas control sera were from individuals proven not to have schistosomiasis clinically and by conventional laboratory findings. Determination of enzyme activities and protein concentration a-Amylase and amyloglucosidase: soluble starch (1% w/v; 11 mL), prepared in 50 mM acetate buffer, pH 5.0, was incubated with either soluble a-amylase (917.5 mg/mL; 7 mL) or amyloglucosidase (100 mg/mL; 100 mL) at 40°C. Samples (200 mL) were withdrawn at appropriate times and mixed to 3,5-dinitrosalicylic acid (DNSA) reagent (2 mL). These mixtures were heated at 100°C for 10 min and then read at 570 nm. On the other hand, PVA-glutaraldehyde beads containing either aamylase (2 g) or amyloglucosidase (1.5 g) were incubated with the soluble starch (20 mL) with stirring. Samples (1 mL) were withdrawn and immediately centrifuged. Aliquots (200 mL) of the supernatants were mixed to DNSA (2 mL) and proceeded as above. Xanthine oxidase: xanthine (200 mM; 10 mL), prepared in 0.1 M phosphate buffer, pH 8.0, containing 0.1 mM EDTA, was incubated with the xanthine oxidase PVAglutaraldehyde beads (0.5 g) at 25°C with stirring. Aliquots (1 mL) were withdrawn at time intervals, centrifuged (2,500 3 g) and the supernatants read at 293 nm (Hitachi 3200 spectrophotometer). Protein concentration: determined according to Lowry (1951). Enzyme Linked Immunosorbent Assay (ELISA) According to Araujo et al. (1996).

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Results and discussion Since it was accidentally discovered (Haehnel and Herrmann; 1924), PVA has become one of the most studied polymer in the literature. Applications ranging from water insoluble textile fibre to support for biomaterial immobilisation such as heparin (Rollason and Sefton, 1986), catalase (Tarhan, 1990) and drugs (Thanoo et al., 1992) have been proposed. The reason for this seems to be the fact that PVA can be industrially produced rather cheaply, it is non-toxic and can form membranes with large surface areas suitable for enzyme reaction (Kozhukharova et al., 1988). In our laboratory, a polymer network based on PVA-

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glutaraldehyde semi-interpenetrating network was prepared (De Souza et al., 1994). The infrared analysis showed that when the samples were prepared below the stoichiometric relations, some carbonyl groups remained free and it increased as the glutaraldehyde concentration increased. From these results high swelling discs with free carbonyl groups able to covalently bind amine groups from biomaterials were synthesised. PVA and the most probably mechanism reaction with glutaraldehyde are presented below: [– CH – CH2 – CH – CH2 – CH – CH2 – CH – CH2 –]n OH

OH

OH

OH

Isostatic stereoisomeric form of polyvinyl alcohol H+

HOC–(CH2)3 –COH Glutaraldehyde

[– CH – CH2 – CH – CH2 – CH – CH2 – CH – CH2 –] O

O

OH

OH

C H

(CH2)3 CHO

Glutaraldehyde reaction with PVA via acetal mechanism under acid catalysis.

Glutaraldehyde as a bifunctional molecule can react either to other PVA chain yielding a polymer network or to available NH2 group from proteins. Goldstein and Menecke (1976) suggested that the glutaraldehyde reaction most probably involves conjugated addition of protein aminogroups to ethylene double bonds of a,bunsaturated glutaraldehyde oligomers, since the linkages formed between the protein and glutaraldehyde are irreversible and survive extremes of pH and temperature. Therefore, the possibility of Shiff’s base (aldiimine) formation has been kept to a minimum due to the reaction’s reversibility in aqueous media, particularly at low pH values. Table 1 shows the specific activity retention of a-amylase, amyloglucosidase and xanthine oxidase after covalent immobilisation on PVA-glutaraldehyde beads, here used as models to test this support. According to these results one can observe the high percentage of specific activity retained for xanthine oxidase (72.3%). The Michaelis constant estimated for this preparation was estimated as 24 ± 4 mM using xanthine as substrate. This value is slightly higher than that obtained for the soluble enzyme (16 ± 2 mM).

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Table 1 Retention of specific activity of immobilised enzymes on pva-glutaraldehyde beads Enzyme

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Specific activity (units/mg protein)

Xanthine oxidase Soluble Immobilised a-Amylase Soluble Immobilised Amyloglucosidase Soluble Immobilised

Retention (%)

46.0 33.2

100.0 72.3

65.3 1.1

100.0 1.6

6.4 0.1

100.0 1.4

On the other hand, poor results were observed for the carbohydrases specific activity retentions. These results can be attributed to the high molecular weight substrate (starch) used to assay their activities. However, maximal hydrolysis of starch can be achieved using the a-amylase derivative as can be seen in Fig. 1. Araujo et al. (1996) and Carvalho et al. (1996) have already proved that antigens can be covalently linked to PVA-glutaraldehyde disc using a F1 antigen extracted from Yersinia pestis. Here, soluble proteins of adult worms of Schistosoma mansoni were covalently linked to this modified polymer and Fig. 2 shows the

relationship between the amount of fixed antigen and the results obtained by ELISA. A hyperbolic curve was attained and the plateau means that saturation of antigen-antibody complex was achieved under these experimental conditions. According to this result antigen amount of 1.3 mg is required to set up an ELISA which is smaller than that using PVC microplates (5 mg). The optical density means (± standard deviations) for the ELISA carried out in sera from schistosomotic and healthy individuals (n = 16) were equal to 0.378 ± 0.090 and 0.017 ± 0.014, respectively. These two means are significantly different at the 0.05 level provided that the independent t-test showed a p