POLYDOPAMINE-IMMOBILIZED CONCANAVALIN A EXIBITS SELECTIVE BINDING OF RNASE B GLYCOFORMS

 

Todd A. Morris and Michael J. Tarlov

†Chemical Science and Technology Laboratory, NIST Gaithersburg , MD 20899.

 

Nearly ĺ of all protein drugs are glycoproteins, where complex oligosaccharides, or glycans, are attached to the polypeptide backbone.† The composition and structure of these glycans are known to strongly influence the safety and efficacy of the protein therapeutic.† Thus, measurement of protein glycosylation is important in the manufacture and quality control of these medicines.†† An emerging method for glycoanalysis is the use of lectin arrays.† Lectins are proteins that can recognize and bind specific oligosaccharides.† Critical to the performance of these arrays is the immobilization of the lectin molecules.† Here, I report the use of polydopamine films for linking lectins to surfaces for the construction of lectin arrays.† Polydopamine is formed by the autoxidation of dopamine under aerated and alkaline conditions and will adsorb to virtually any surface. In this study, polydopamine (pDA) was used to modify gold, indium, aluminum, and iridium surfaces and promote the adhesion of the a-mannose-specific lectin Concanavalin A (Con A). The activity of the surface-bound lectin was demonstrated with the a-mannose-presenting glycoprotein Ribonuclease B (RNAse B). Surface plasmon resonance spectroscopy (SPRS) was used to demonstrate the selectivity affinity of Con A for RNAse B. By Scatchard plot analysis of the SPRS data, a dissociation constant (KD) of 4 mM was determined for RNAse B and immobilized Con A. In addition, surface-MALDI-TOF MS experiments revealed that the affinity of Con A/pDA for the glycoforms of RNAse B is significantly affected by slight variations in oligosaccharide structure or composition.

 

 

 

 

 

 


 

CATEGORY: Chemistry

 

 

Mentorís Name: Michael J. Tarlov

CSTL, Process Measurements Division

Bldg 221/B328 MS 8362

Tel: 301-975-2563

Fax: 301-975-2643

todd.morris@nist.gov

 

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