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New high-performance liquid chromatography assay yeglycosyltransferases yakavakirwa pakubuda ne anthranilic acid uye fluorescence kuonekwa.

Miyedzo yakagadziridzwa ichishandisa yakasarudzika kemikari ye2-aminobenzoic acid (2AA; anthranilic acid, AA) yekuyera zviitiko zveese β1-4 galactosyltransferase (GalT-1) uye α2-6 sialyltransferase (ST-6) nepamusoro-inoshanda mvura. chromatography (HPLC) nekuongorora kwefluorescence (Anumula KR. 2006. Kufambira mberi mu fluorescence derivatization nzira dzepamusoro-performance liquid chromatographic analysis ye glycoprotein carbohydrates. Anal Biochem. 350: 1-23).N-Acetylglucosamine (GlcNAc) uye N-acetyllactosamine yakashandiswa sevanobvuma uye uridine diphosphate (UDP) -galactose uye cytidine monophosphate (CMP) -N-acetylneuraminic acid (NANA) sevapi veGalT-1 uye ST-6, maererano.Zvigadzirwa zveEnzymatic zvakanyorwa mu situ neAA uye zvakapatsanurwa kubva kune substrates paTSKgel Amide 80 column vachishandisa zvakajairika-chikamu mamiriro.Enzyme units dzakatemerwa kubva kunzvimbo dzepamusoro nekuenzanisa neyakafanana yakatorwa zviyero Gal-β1-4GlcNAc uye NANA-α2-6 Gal-β1-4GlcNAc.Linearity (nguva uye enzyme concentration), kunyatsojeka (intra- uye interassay) uye reproducibility yezviyero zvakasimbiswa.Iwo maedzo akawanikwa achibatsira mukutarisa zviitiko zve enzyme panguva yekuzviparadzanisa nevamwe uye kucheneswa.Iwo ma assays aive akanyanya kunzwa uye akaitwa akaenzana kana nani pane echinyakare radioactive shuga-based zviyero.Iyo fomati yeassay inogona zvakare kushandiswa kuyera chiitiko chezvimwe zvinotamiswa, chero izvo zvinogashira makhahydrates zvine magumo ekudzikisa ekunyora.Muedzo wevanogamuchira glycoprotein wakagadzirwa uchishandisa IgG.Ipfupi HPLC profiling nzira yakagadziridzwa yekuparadzaniswa kweIgG glycans (biantennary G0, G1, G2, mono- uye disialylated), iyo yakafambisa kutsunga kweGalT-1 uye ST-6 zviitiko nekukurumidza.Uyezve, iyi nzira yekunyora inofanirwa kuratidza kubatsira pakutarisa shanduko muIgG glycans mumakiriniki marongero.