Monoclonal antibodies as probes for monitoring the denaturation process of bovine beta-lactoglobulin.
Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.
Kaminogawa
S
S
Department of Agricultural Chemistry, University of Tokyo, Japan.
Shimizu
M
M
Ametani
A
A
Hattori
M
M
Ando
O
O
Hachimura
S
S
Nakamura
Y
Y
Totsuka
M
M
Yamauchi
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D006358:Hot Temperature; D007782:Lactoglobulins / Q000032:analysis / Q000276:immunology*; D010455:Peptides / Q000032:analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007782:Lactoglobulins; D010455:Peptides