34监测蛋白质 Monitoring proteins Using Molecular Beacons To Probe Molecular Interactions between Lactate Dehydrogenase and Single-Stranded DNA Xiaohong Fang, Jianwei Jeff Li, and Weihong Tan Department of Chemistry and UF Brain Institute, University of Florida, Gainesville, Florida 32611 The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB)DNA probe for the analysis of an enzyme, lactate dehydrogenase(LDH), and for the investigation of its properties of binding with single-stranded DNA Molecular beacons are single stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. the interaction between LDh and Mb has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of M B/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1: 1, and the binding constant is 1.9x10-7M-1. We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzy mes Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNAinteraction studies MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real- time measurements Analytical Chemistry, Vol. 72, No. 14, July 15, 20003.4 监测蛋白质(Monitoring proteins) The interactions between two key macromolecular species, nucleic acids and proteins, control many important biological processes. There have been limited effective methodologies to study these interactions in real time. In this work, we have applied a newly developed molecular beacon (MB) DNA probe for the analysis of an enzyme, lactate dehydrogenase (LDH), and for the investigation of its properties of binding with single-stranded DNA. Molecular beacons are single￾stranded oligonucleotide probes designed to report the presence of specific complementary nucleic acids by fluorescence detection. The interaction between LDH and MB has resulted in a significant fluorescence signal enhancement, which is used for the elucidation of MB/LDH binding properties. The processes of binding between MB and different isoenzymes of LDH have been studied. The results show that the stoichiometry of LDH-5/MB binding is 1:1, and the binding constant is 1.910-7 M-1 . We have also studied salt effects, binding sites, temperature effects, pH effects, and the binding specificities for different isoenzymes. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has a signal transduction mechanism built within the molecule and can thus be used for the development of rapid protein assays and for real-time measurements