IONTOSORB - Bead Cellulose Derivatives
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Immobilization Enzymes by Covalent Bond

Enzyme Immobilization by Diazotation Method

This method of covalent binding is based on the reaction of aromatic primary amines with HNO₂ forming diazo-salts. Consequently these diazo-salts are coupled with proper groups in reacting proteins (especially with phenolic groups of tyrosine and imidazole groups of histidine).

For the IONTOSORB AV the following procedure was used :

To 1 g support Iontosorb AV in 100 ml HCl (c = 2.5 mol/l) there was added gradually 100 ml water-solution of NaNO₂ (concentration 2 %) at 0 ^oC. After 15 min (reaction time) the activated support was separated and rinsed 4times with 100 ml phosphate buffer (c = 0.1 mol/l) at the same pH value as during immobilization. To a sucked off support 10 ml enzyme solution was added. The enzyme solution contained GOD, KAT - 10 mg in 10 ml phosphate buffer (c = 0.1 mol/l) at pH 7.2 or CHT - 20 mg in 10 ml phosphate buffer (c = 0.1 mol/l) at pH 7.8. The mixture was shook for 24 hours at 4 ^oC.   The obtained conjugate was sucked off and rinsed in the alternate way with 150 ml phosphate buffer (c = 0.1 mol/l) and 80 ml NaCl (c = 1 mol/l) in the same buffer with the adequate pH value. Immobilized enzymes were saved at 4 ^oC covered with phosphate buffer.

alpha-Chymotrypsine (CHT), Glucoseoxidase (GOD) and Catalase (KAT)

The results of immobilization by diazotation method are given in the following table:

MS = moist support                                 DS = dry support