New Heterobifunctional Cross-Linking Reagents for Protein Conjugation, N-(Bromoacetamido-n-alkanoyloxy)succinimides(Analytical,Chemical)

ZAITSU K., OHNISHI MIKIO, HOSOYA HIROYUKI, SUGIMOTO HIROYUKI, OHKURA YOSUKE

Five heterobifunctional reagents, N-(bromoacetamido-n-alkanoyloxy)succinimides having a glycine or ω-amino acid (β-alanine, γ-aminobutyric acid, δ-aminovaleric acid or ε-aminocaproic acid) residue, were synthesized. Bromoacetamido-η-alkanoyl groups could be introduced into horseradish peroxidase and hen egg-white lysozyme and its reduced and S-3-(trimethylated amino)propylated product by using the reagents. The number of bromoacetamido-η-alkanoyl groups introduced into the proteins could be successfully estimated from the amount of glycine or ω-amino acid (non-protein amino acid) produced from the introduced groups by acid hydrolysis. N-(β-Bromoacetamido-η-propionoyloxy)succinimide, one of the reagents, was examined as a reagent for the preparation of horseradish peroxidase-insulin conjugate. A single β-bromoacet-amido-η-propionoyl group was first introduced into Gly^<A1>, Phe^<B1>-dicitraconylinsulin through the ε-amino group of its Lysine^<B29> residue and the product was subjected to decitraconylation with dilute acetic acid to obtain Lys^<B29>-(β-bromoacetamido-η-propionoyl)insulin. The insulin was reacted with thiolated horseradish peroxidase to give peroxidase-insulin conjugate (molar ratio of 1:1).

New Heterobifunctional Cross-Linking Reagents for Protein Conjugation, N-(Bromoacetamido-n-alkanoyloxy)succinimides(Analytical,Chemical)

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