Phenylboronic Acid−Salicylhydroxamic Acid Bioconjugates. 2. Polyvalent Immobilization of Protein Ligands for Affinity Chromatography

• Published in: Bioconjugate chemistry Vol. 12; no. 2; pp. 240 - 250
• Main Authors: Wiley, Jean P, Hughes, Karin A, Kaiser, Robert J, Kesicki, Edward A, Lund, Kevin P, Stolowitz, Mark L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01-03-2001
• Subjects: Alkaline Phosphatase - chemistry; Alkaline Phosphatase - metabolism; Boronic Acids - chemistry; Chromatography, Affinity - methods; Enzyme Stability; Enzymes, Immobilized - chemistry; Horseradish Peroxidase - chemistry; Horseradish Peroxidase - metabolism; Humans; Hydrogen-Ion Concentration; Immunoblotting; Immunoglobulin G - immunology; Immunoglobulin G - metabolism; Molecular Structure; Protein Binding; Salicylamides - chemistry; Sepharose - chemistry
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc000086l

Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-{N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl}propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-{[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino}hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 μmol of SHA/mL of gel), PDBA−alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA−AP·SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (≥7 μmol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 μmol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA−αHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.