Affinity capillary electrophoresis applied to the studies of interactions of a member of heat shock protein family with an immunosuppressant.
The bioaffinity of receptor-ligand interactions is investigated by determining the binding constant (association constant or dissociation constant) of the resulting complex utilizing affinity capillary electrophoresis (ACE). The ACE binding assay was established with a potent immunosuppressant, deoxyspergualin (DSG), that binds specifically to Hsc70, a constitutive or cognate member of heat shock protein 70 (Hsp70) family. Quantitative determination of binding constants under different running buffer systems provide comparative results. The association constants for the interaction between Hsc70 protein and DSG were found to be 5.7 x 10(4) M-1 in a buffer with pH 6.95 and 6.3 x 10(4) M-1 in a buffer with pH 5.30 (or corresponding dissociation constants, 18 and 16 microM, respectively) based on Scatchard analyses. Binding of DSG to a synthetic peptide, SINPDEAVAYGAAVQAAILSGDK, one of the DSG-binding fragments found from tryptic digest of Hsc70 protein, provides further detailed information for the understanding of Hsc70 binding domain. The applicability of using coated capillaries was also evaluated for probing Hsc70-DSG interaction.
Liu
J
J
Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492.
Volk
K J
KJ
Lee
M S
MS
Kerns
E H
EH
Rosenberg
I E
IE
D000595:Amino Acid Sequence; D001665:Binding Sites; D002021:Buffers; D002197:Capillary Action; D002352:Carrier Proteins / Q000378:metabolism*; D004586:Electrophoresis / Q000379:methods*; D006146:Guanidines / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007166:Immunosuppressive Agents / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D002021:Buffers; D002352:Carrier Proteins; D006146:Guanidines; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D007166:Immunosuppressive Agents; D010446:Peptide Fragments; D014357:Trypsin; C037258:gusperimus