Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography.
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 micromol 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 (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman 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.
Wiley
J P
JP
Prolinx, Inc., 22322 20th Avenue SE, Bothell, Washington 98021, USA.
Hughes
K A
KA
Kaiser
R J
RJ
Kesicki
E A
EA
Lund
K P
KP
Stolowitz
M L
ML
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000378:metabolism; D001897:Boronic Acids / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D015394:Molecular Structure; D011485:Protein Binding; D012457:Salicylamides / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry*
D001897:Boronic Acids; D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D012457:Salicylamides; C005703:salicylhydroxamic acid; D012685:Sepharose; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; C010686:benzeneboronic acid