An easy method for the determination of initial rates.
Ontology highlight
ABSTRACT:
SUBMITTER: Waley SG
PROVIDER: S-EPMC1162697 | biostudies-other | 1981 Mar
REPOSITORIES: biostudies-other
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Project description:The investigation of the mechanism of protein folding is complicated by the context dependence of the rates of intramolecular contact formation. Methods based on site-specific labeling and ultrafast spectroscopic detection of fluorescence signals were developed for monitoring the rates of individual subdomain folding transitions in situ, in the context of the whole molecule. However, each site-specific labeling modification might affect rates of folding of near-neighbor structural elements, and thus limit the ability to resolve fine differences in rates of folding of these elements. Therefore, it is highly desirable to be able to study the rates of folding of two or more neighboring subdomain structures using a single mutant to facilitate resolution of the order and interdependence of such steps. Here, we report the development of the "Transfer-Quench" method for measuring the rate of formation of two structural elements using a single triple-labeled mutant. This method is based on Förster resonance energy transfer combined with fluorescence quenching. We placed the donor and acceptor at the loop ends, and a quencher at an α-helical element involved in the node forming the loop. The folding of the triple-labeled mutant is monitored by the acceptor emission. The formation of nonlocal contact (loop closure) increases the time-dependent acceptor emission, while the closure of the labeled helix turn reduces this emission. The method was applied in a study of the folding mechanism of the common model protein, the B domain of staphylococcal protein A. Only natural amino acids were used as probes, and thus possible structural perturbations were minimized. Tyr and Trp residues served as donor and acceptor at the ends of a long loop between helices I and II, and a Cys residue as a quencher for the acceptor. We found that the closure of the loop (segment 14-33) occurs with the same rate constant as the nucleation of helix HII (segment 33-29), in line with the nucleation-condensation model.
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Project description:BackgroundThe objectives of this study were to develop an easy and rapid method for measuring gene expression in a small number of cells by real-time PCR without RNA extraction and purification, and to use this method to determine more precisely IGF-I gene expression in the cumulus cells surrounding oocytes.MethodsFirst, after small numbers of cumulus cells were lysed in cell lysis buffer, they were digested with various concentrations of DNase I for different periods at 37 degrees C to determine the optimal conditions for digestion of genomic DNA in the lysate. Since nonspecific amplification was liable to occur when the non-purified RT product of the cell lysate was used for real-time PCR with the given primers, the optimal conditions for Mg2+ and annealing temperature were well investigated. Further, to create the same conditions as in the actual sample reaction for measurement by real-time PCR, RT-minus product was added to the reaction mixture of the standard curve, and then the amplification efficiency was assessed. Next, IGF-I gene expression in cumulus cells collected from cumulus oocyte complexes (COCs) every 4 h during maturation was determined using the developed method.ResultsThe optimal conditions for measuring gene expression using the cell lysate from a small number of cells were as follows: incubation of the cell lysate with 0.16 U/microL DNase I with 10 U/microL for 30 min, an Mg concentration of 1.5 mM for amplification of target gene by real-time PCR using RT-product of the cell lysate. When the RT-minus products added to the reaction mixture for the standard curve, which was prepared for purified 18SrRNA plasmid, the PCR efficiency was similar between the sample and the standard. The IGF-I gene expression in the cumulus cells was elevated up through the first 8 h of the culture and then declined gradually by the end of maturation, with the maximal gene expression (778-fold) seen at 8 h.ConclusionIt can be concluded that the method developed here, in which equivalent to cumulus cells collected from 0.03-0.075 COCs were employed per reaction, permits rapid and easy determination of target gene expression in a limited number of cells using real-time PCR without RNA extraction.
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Project description:Excellent estimations of initial rates can be obtained from plots of delta P/t versus product formed (where P is the instantaneous concentration of the product). delta P/t is the chord from P0,t0 to P,t on an ordinary P-versus-t plot. When the chord is plotted as a function of product, the intercept at P0 of the resulting curve is necessarily dP/dt0. This curve approximates to a straight line extremely closely in all cases tested thus far. If delta P/t versus product is calculated from the integrated rate equation for a first-order reaction, and if a straight line is fitted through points representing the first 50% of the reaction, the discrepancy between the true initial rate and dP/dt0 estimated from the plot is 0.68%. For the most common form of the integrated rate equation for catalysed reactions the discrepancy varies between 0 and 0.90%. Because of the complexities of the integrated rate equations, catalysed second-order reactions have not been evaluated directly; uncatalysed reactions have been done instead. For a reaction with one reactant and two products, the discrepancy varies from 0.68 to 2.02%. For two reactants and one product, it varies from 0 to 0.68%; for two and two, 0 to 2.02%. The larger discrepancies occur only when unfavourable equilibrium constants are being overcome by the initial conditions.
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