Résumé
BACKGROUND:Measurement of cardiac troponin plays an important role in diagnosis of myocardial infarction.OBJECTIVE:To label the rabbit cardiac troponin C(cTnC) by a fluorescent probe 5-iodoaccetamidofluorescein(5-IAF),and to observe whether the 5-IAF can be used to study the interaction between cTnC and other contractile regulatory proteins.DESIGN,TIME AND SETTING:A randomized control experiment was performed at Department of Human Anatomy,Guangzhou Medical College,from January 2002 to December 2005.MATERIAL:Adult rabbits were provided by Experimental Animal Center of Guangzhou Medical College.METHODS:The rabbit cTnC DNA fragment was prepared with RT-PCR method.This gene fragment was cloned to pET expression vector by gene recombination technology.The site-directed mutagenesis were used to produce a mutant containing single cysteine at position 84 by replacing Cys35 with Ser,cTnC(C35S).The cTnC(C35S) was labeled by 5-IAF and 2-(4'-(iodoacetamido) anilino) naphthalene-6sulfonic acid(IAANS),Respectively.And then,the fluorescence emission(steady-state and time-resolved) was performed.MAIN OUTCOME MEASURE:The fluorescence properties of 5-IAF-labeled cTnC(C35S) and IAANS-labeled cTnC(C35S).RESULTS:The excitation of apo-cTnC(C35S)IAF was performed at 491 nm,and the emission peak was at 520 nm.Saturation of cTnC(C35S)IAF with Mg led to a 35% decrease in fluorescence intensity.Another 35% decrease with a 3 nm-blue shift was seen as the protein was saturated with Ca.The two-phase transitions of fluorescence emission from IAANS-labeled cTnC in response to Mg and Ca did not appear in fluorescence emission of 5-IAF-labeled cTnC.However,the Ca-induced conformational change in cTnC remained unchanged no matter which probe was used.Ca titration experiments showed that binding parameters derived from the fluorescence emission of the two probes were comparable.CONCLUSION:5-IAF is an appropriate probe that can be used to study the interaction between cardiac troponin C and other contractile regulatory proteins.