Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(III) complex.
A complex of an independent L chain from anti-mesotetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90 degrees C). Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment. The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone. Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115-146) and hydrophilic region (147-189) in CL are important for thermostability and activity, respectively. Tertiary structure of L-zyme was predicted by AbM. Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity. Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively. Distance between proximal His and distal His in L-zyme is 9.09 A, whereas that of ARP is 7.8 A.
Kohda
K
K
Department of Biotechnology, Faculty of Engineering, Osaka University, Suita, Yamada-oka, Japan.
Kakehi
M
M
Ohtsuji
Y
Y
Tagaki
M
M
Imanaka
T
T
D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011166:Porphyrins / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D017151:Antibodies, Catalytic; D000911:Antibodies, Monoclonal; D007147:Immunoglobulin Light Chains; D011166:Porphyrins; D011994:Recombinant Proteins; C018395:tetracarboxyphenylporphine; D007501:Iron; D010544:Peroxidases