Pseudo-affinity chromatographic approach to probe heterogeneity in buffalo pituitary luteinizing hormone: probable pseudolectin-like behavior of immobilized Cibacron Blue 3GA.
The alpha (α) and beta (β) subunits of buffalo pituitary luteinizing hormone (LH) were chromatographed on Cibacron Blue 3GA agarose and their immunoreactivity was quantitated using anti-α and anti-β anti sera. Subsequent analyses showed α subunits were relatively more hydrophilic than β subunits. Further, the naturally occurring free α and β subunits were more hydrophobic than their native counterparts which were dissociated and isolated from heterodimeric LH. The lesser sugar content in freely occurring α and beta subunits may be attributed for increased hydrophobicity and consequent upon the existence of their uncombined free forms. In order to ascertain putative sugar-dye interaction, crude LH carrying free subunits, pure LH, and non-glycosylated recombinant β subunit of LH were loaded separately on Cibacron Blue. Methyl mannoside was able to elute 33% of the bound protein in case of crude and pure LH, whereas there was little (3%) elution in case of recombinant LH β subunit. This study suggests a compositional heterogeneity in free and native subunits of LH from the buffalo pituitary. In addition, our findings reveal the pseudolectin-like behavior of Cibacron Blue.
Arora
Taruna
T
Department of Zoology, University of Delhi, Delhi, India.
Vashistha
Nidhi
N
Chaudhary
Rajesh
R
Muralidhar
Kambadur
K
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002020:Buffaloes / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D007986:Luteinizing Hormone / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014227:Triazines / Q000737:chemistry / Q000378:metabolism*
D000906:Antibodies; D021122:Protein Subunits; D011994:Recombinant Proteins; D014227:Triazines; C012842:Cibacron Blue F 3GA; D007986:Luteinizing Hormone; D000645:Ammonium Sulfate