Analysis of thermal stability of soya globulins using monoclonal antibodies.
The epitopes of two monoclonal antibodies (Mabs), one raised to soya glycinin (IFRN 0025) and one to beta-conglycinin (IFRN 0089), have been defined. The epitope of 0025 corresponds to residues 86-104 of the acidic polypeptide of glycinin A1aB1b and lies at the C terminus of the proteolytic intermediate known as glycinin-T, whilst that of 0089 corresponds to residues 78-84 in the acidic extension present in the alpha' subunit of beta-conglycinin. As the Mabs bind to synthetic peptides corresponding to the epitope regions both epitopes can be considered as being continuous in nature. The regions recognised correspond to surface loops, which are probably flexible in nature. Both Mabs were used to investigate thermally induced conformational changes in soya globulins. Thermally treated glycinin was recognised more strongly than native protein, possibly due to disruption of the disulphide bond joining the acidic and basic polypeptides. Disruption could increase epitope accessibility, as could the conformational changes associated with denaturation. The binding of anti-beta-conglycinin Mab 0089 was unaffected by heating, suggesting that its epitope remains on the surface of the aggregates formed on heating. This study demonstrates that Mabs with defined specificities can be sensitive probes for monitoring local conformational changes within a protein molecule during thermal denaturation.
Department of Biochemistry, Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK.
L
L
Huang
E N
EN
Mills
J M
JM
Carter
M R
MR
Morgan
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005916:Globulins / Q000737:chemistry* / Q000276:immunology; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011489:Protein Denaturation; D055314:Seed Storage Proteins; D030262:Soybean Proteins; D013025:Glycine max / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D000939:Epitopes; D005916:Globulins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max; C011474:glycinin