I-Fluorescence resonance energy transfer (FRET)
Ukudluliswa kwamandla kwe-Fluorescence resonance (FRET) kuyinqubo yokudlulisa amandla angasebenzisi imisebe lapho amandla ombuso ajabulile adluliselwa kumamukeli wesimo esijabulile ngokusebenzisana kwemibhangqwana kagesi yama-intermolecular.Le nqubo ayibandakanyi ama-photon ngakho-ke ayiyona imisebe.Lokhu kuhlolwa kunezinzuzo zokushesha, ukuzwela nokulula.
Udayi osetshenziswe ekuhlolweni kwe-FRET ungafana.Kodwa emisebenzini eminingi, odayi abahlukene basetshenziswa ngempela.Kafushane, ukudluliswa kwamandla okukhanya kwe-resonance ukudluliselwa kwepheya lama-dipoles ukusuka kumnikeli (udayi 1) kuya kumamukeli (udayi 2) lapho iqembu labanikezeli lijabule.Ngokuvamile, i-spectrum ekhishwayo yeqembu le-Donor fluorophore igqagqana ne-spectrum yokumuncwa yeqembu le-Acceptor."Lapho ibanga eliphakathi kwama-fluorophore amabili lifanelekile (10 — 100 A), ukudluliswa kwamandla e-fluorophore ukusuka kumnikeli kuya kumamukeli kungabonwa."Indlela yokudlulisa amandla incike esakhiweni samakhemikhali se-receptor:
1. Iguqulwa ibe ukudlidliza kwamangqamuzana, okungukuthi, ukukhanya okukhanyayo kokudluliswa kwamandla kuyanyamalala.(I-receptor iyisicimi sokukhanya)
2. Ukukhishwa kunamandla kakhulu kunesamukeli ngokwaso, okuholela ekushintsheni okubomvu ku-spectrum yesibili ye-fluorescence.”(Ama-receptors angama-emitters akhanyayo).
Iqembu labanikelayo (EDANS) kanye nofuzo olwamukelayo (DABCYL) zixhunywe ngokufanayo ku-substrate yemvelo ye-HIV protease, futhi lapho i-substrate inganqanyulwa, i-DABCYL ingacisha i-EDANS bese ingatholakali ku-fluorine.Lapho kunqanyulwa i-HIV-1 protease, i-EDANS ayisacinywa yi-DABCYL, futhi i-EDANS luciferases ingatholwa kamuva.Ukutholakala kwama-protease inhibitors kungagadwa izinguquko ku-fluorescence intensity ye-EDANS.
Ama-peptide angu-FRET angamathuluzi alula okutadisha ukungacaciswa kwe-peptidase.Njengoba inqubo yokusabela kwayo ingaqashelwa ngokuqhubekayo, inikeza indlela elula yokuthola umsebenzi we-enzyme.I-sheen ekhiqizwa ngemva kwe-hydrolysis yamabhondi e-peptide ngumnikeli/umamukeli inikeza isilinganiso somsebenzi we-enzyme ekugxilweni kwe-nanomolar.Uma i-peptide ye-FRET iphelele, ibonisa ukunyamalala kungazelelwe kwe-flash yangaphakathi, kodwa uma noma iyiphi ibhondi ye-peptide emelene nomnikezeli/umamukeli iphuka, ikhulula ukukhanya, okungatholakala ngokuqhubekayo futhi umsebenzi we-enzyme ungabe usubalwa.
Isikhathi sokuthumela: Aug-14-2023