Simultaneous estimation of the association constants of glycoprotein glycoforms to a common protein by capillary electrophoresis.
The efficacy of our capillary electrophoresis method for simultaneous estimation of the association constants of glycoprotein glycoforms to a common target protein was demonstrated using ribonuclease and ovalbumin glycoforms as glycoform models and Lens culinaris agglutinin (LCA) as a protein model. The ribonuclease glycoforms were fairly well separated in the absence of LCA at pH 5.8, but the peaks were retarded without any change of separation profile in the presence of LCA, the retardation becoming greater as LCA concentration increased. The estimated values of apparent association constant (K(a)) were at the 10(6)M(-1) level for all the ribonuclease glycoforms, and there was no significant difference among glycoforms. The high-mannose-type N-glycans released from a mixture of ribonuclease glycoforms gave lower values of K(a) at the 10(4)-10(5)M(-1) level to the same protein, and the glycans having a larger number of the mannose residue gave larger K(a) values. These results imply that the glycan moiety in this glycoprotein might contribute to its binding to the protein, but the polypeptide core played the major role. In contrast, ovalbumin glycoforms gave poorly resolved peaks in the absence of LCA, but they were separated into several peaks in the presence of LCA, which were tentatively assigned based on the knowledge of affinity to this lectin, and K(a) values were estimated simultaneously. The estimated K(a) values were smaller than those of the ribonuclease glycoforms, suggesting the major role of the N-glycan moiety. Thus, capillary electrophoresis allowed simultaneous estimation of K(a) values under common conditions using small amounts of glycoform mixtures and proteins without prior isolation and purification. Comparison of the obtained values will provide useful information on the glycan structure-affinity correlation.
Uegaki
Kana
K
Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan.
Taga
Atsushi
A
Akada
Yoshinobu
Y
Suzuki
Shigeo
S
Honda
Susumu
S
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002645:Chickens; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008358:Mannose / Q000737:chemistry; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000032:analysis / Q000378:metabolism; D037121:Plant Lectins / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000032:analysis / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000032:analysis / Q000378:metabolism; D012260:Ribonucleases / Q000032:analysis / Q000378:metabolism
D006023:Glycoproteins; D037121:Plant Lectins; D011134:Polysaccharides; D020033:Protein Isoforms; C033619:lentil lectin; D010047:Ovalbumin; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic; D008358:Mannose