title: Boronic acid lectin affinity chromatography (BLAC). 3. Temperature dependence of glycoprotein isolation and enrichment.
abstract: In this paper, the effect of temperature is investigated on the performance of glycoprotein enrichment by boronic acid lectin affinity chromatography (BLAC). Wheat germ agglutinin and m-aminophenyl boronic acid containing stationary phases were evaluated individually and in a mixed mode using an automated liquid handling robot with an integrated 96-well plate temperature controller. Glycoaffinity enrichment of the model proteins of ribonuclease B and trypsin inhibitor was investigated in the presence of the non-glycosylated proteins of myoglobin (neutral) and lysozyme (basic) at a wide temperature range of 5-65 degrees C. Our results revealed that glycoaffinity micropartitioning at the temperature of 25 degrees C provided the highest recovery rate for glycoprotein enrichment. We have also found that a large amount of lysozyme was present in the elution fractions of the m-aminophenyl boronic acid containing micropartitioning columns due to ion-exchange mechanism occurring between the positively charged protein and the negatively charged stationary phase at the operation pH. On the other hand, at high temperature (65 degrees C), non-specific interactions with the agarose carrier prevailed, evidenced by the presence of myoglobin in the eluate.
authors.affiliation: Horváth Laboratory for Bioseparation Sciences, University of Debrecen, 4032 Debrecen, Hungary.
authors.forename: Marcell
authors.initials: M
authors.lastname: Olajos
authors.forename: Akos
authors.initials: A
authors.lastname: Szekrényes
authors.forename: Peter
authors.initials: P
authors.lastname: Hajos
authors.forename: Doug T
authors.initials: DT
authors.lastname: Gjerde
authors.forename: András
authors.initials: A
authors.lastname: Guttman
mesh_terms: D000818:Animals; D001897:Boronic Acids / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D006023:Glycoproteins / Q000302:isolation & purification*; D007474:Ion Exchange; D037102:Lectins / Q000737:chemistry; D009113:Muramidase / Q000302:isolation & purification; D009211:Myoglobin / Q000302:isolation & purification; D012260:Ribonucleases / Q000302:isolation & purification; D013696:Temperature; D014361:Trypsin Inhibitors / Q000302:isolation & purification; D014909:Wheat Germ Agglutinins / Q000737:chemistry
chemical_list: D001897:Boronic Acids; D006023:Glycoproteins; D037102:Lectins; D009211:Myoglobin; D014361:Trypsin Inhibitors; D014909:Wheat Germ Agglutinins; D012260:Ribonucleases; C029600:ribonuclease B; D009113:Muramidase