Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody is Governed by Chemical Modifications

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2014 Apr 18

PMID 24739391

Citations 21

Authors

Yoko Akazawa-Ogawa

Mizuki Takashima

Young-Ho Lee

Takahisa Ikegami

Yuji Goto

Koichi Uegaki

Yoshihisa Hagihara

Affiliations

Soon will be listed here.

Abstract

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.

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