Imikhiqizo

I-Monogalactopyranosides ye-fluorescein ne-fluorescein methyl ester: i-synthesis, i-enzymatic hydrolysis nge-biotnylated β-galactosidase, kanye nokunqunywa kwe-coefficient yokuhumusha yokusabalalisa

I-Fluorescein monoglycosides (d-galactopyranoside (FMG) kanye ne-d-glucopyranoside) kanye ne-methyl ester (MFMG) yayo ilungiswe kusukela ku-acetobromoglucose/galactose kanye ne-fluorescein methyl ester ngesivuno esihle.Ukuhlolwa kwe-enzymatic hydrolysis (kusetshenziswa i-biotinylated β-galactosidase) kokuphuma kokuphuma kwe-galacto kwenziwe futhi imingcele ye-kinetic ibaliwe.Ukwanda kwe-fluorescence izikhathi eziyi-15-20 kuye kwabonwa ngesikhathi se-hydrolysis.Ukwenyuka komugqa we-fluorescence kuye kwaphawulwa ngesikhathi esifushane kanye nokugcwala okuphansi kwe-substrate, okwenza lezi zinhlanganisela zibe ama-probe awusizo futhi azwelayo kuma-galactosidase.Ubukhulu bevelu ye-Michaelis–Menten constant (Km) ye-MFMG iphakeme kunaleyo ye-FMG ephakamisa ukuthi kube noshintsho olungenzeka lwe-fluorogenic substrate.Inani le-Km le-β-Gal ye-biotinylated ene-FMG liphansi kunalelo le-enzyme yomdabu.Lokhu kuhlola kubonisa ukuhlobana okuphezulu kwe-substrate kwe-enzyme ye-biotinylated uma kuqhathaniswa ne-enzyme yomdabu.Ama-coefficients okusabalalisa okuhumusha akaliwe, kukho kokubili ama-substrates e-fluorogenic nayo yomibili imikhiqizo, kusetshenziswa i-fluorescence correlation spectroscopy.Ama-coefficients okuhumusha okusabalalisa ama-substrates e-fluorogenic kanye nemikhiqizo ye-enzymatic hydrolysis kuye kwalinganiswa ukufana, kububanzi obungu-3.5–4.5 × 10−10 m2 s−1.Ngakho-ke ukuthuthukiswa noma ukuvinjelwa kwe-kinetics ye-enzymatic ngenxa yomehluko ekuhambeni kokuhumusha kwe-substrate nomkhiqizo akubonakali kanjalo.