Modification of allergenicity and immunogenicity of formate dehydrogenase by conjugation with linear mono methoxy poly ethylene glycol: improvement in detoxification of formate in methanol poisoning.

摘要

BACKGROUND: Single bolus intravenous infusion of native formate dehydrogenase (FD), isolated from Candida boidinii was found to eliminate formate, a highly toxic metabolite in methanol poisoning. In order to prevent immunological reactions which might be produced by multiple dosing of formate dehydrogenase and to prolong the serum half life of the enzyme, the N-hydroxysuccinimidyl ester of methoxy polyethylene glycol propionic acid (mPEG-SPA 5000) was conjugated to native formate dehydrogenase. METHOD: PEGylation reactions were run at 20 degrees C for 30 min in a reaction buffer (0.2 mol/l sodium phosphate buffer, pH 8.3). The PEGylated molecules were purified from unreacted PEG with Amicon Ultra-4 (10 K) and by Sephacryl S-300 HR gel-filtration chromatography. Unreacted formate dehydrogenase molecules were removed by DEAE Sepharose FF anion-exchange chromatography. PEG-FD enzyme molecules obtained from reacting ratio of FD/PEG of 1/40 had an enzyme activity of 68% of unmodified enzyme. Immunogenicity of PEGylated and native enzyme was evaluated by ELISA. Allergenicity was evaluated by active systemic anaphylaxis and passive cutaneous anaphylaxis tests. In vivo efficacy of PEG-FD or native FD was comparatively evaluated by single intravenous administration of PEG-FD or native FD in folate deficient methanol intoxicated albino rats along with Carbicarb buffer infusion. Methanol and formate were estimated at specific time points respectively with HPLC and fluorescence spectrophotometer. RESULT: PEG-FD had comparatively longer half life and lower immunogenicity than native FD. PEG-FD had better in vivo efficacy than native FD in eliminating the formate. CONCLUSION: Conjugation of mPEG-SPA 5000 with native FD reduces its immunogenicity and increases its efficacy in detoxification of formate in methanol poisoning.