An interference of fluorescence derived

An interference of fluorescence derived from biological samples is also an important CZC-25146 to be addressed in the fluorescent analysis for biological samples. Advanced glycation endproducts (AGEs) are representative endogenous fluorescent substances that are widely distributed in various organs as well as plasma. The amount of AGEs would be varied by aging and diabetes mellitus, and it has been shown that AGEs show emission at 440 nm when excited at 370 nm , . To overcome the problem, Alexa Fluor 555 (A555; Invitrogen Life Technologies, Carlsbad, CA, USA), which has longer wavelengths of excitation and emission at 545 and 565 nm, respectively, than those of AGEs, was employed and then incorporated into K18C/D140H to yield K18C/D140H–A555. The procedures for fluorescent dye labeling were described in our previous study . The fluorescence quenching of K18C/D140H–A555 depending on the hemin concentration was measured to confirm whether K18C/D140H–A555 functioned as a heme sensor. The heme titration was performed at 25 °C against 1 nM K18C/D140H–A555 in 20 mM potassium phosphate buffer (pH 7.4) in a measurement cuvette, and fluorescence spectra were recorded during titration. The fluorescence spectra of K18C/D140H–A555 exhibited characteristic emissions at 565 nm when excited at 545 nm (A). Even at low concentrations of hemin (0.2–1.6 nM), the sensor showed a good response to heme, suggesting that the sensor can detect at least 0.2 nM heme. The fluorescent emission remarkably decreased and was quenched almost completely after the addition of an equimolar and excess amount of hemin (>10 nM). To evaluate the binding affinity of K18C/D140H–A555 for heme, we defined the – value, where is the initial intensity of the emission maxima and is the intensity of the emission maxima in the presence of hemin. The – value was plotted against the hemin concentration, and the values were then estimated by nonlinear curve fitting as described previously (A, inset) . This analysis indicated that K18C/D140H–A555 had 1:1 binding stoichiometry with heme. The estimated value was 0.13 ± 0.02 nM, which was 10- to 20-fold smaller than the values for previously reported sensors—K18Cs labeled with Alexa Fluor 350 (A350; Invitrogen Life Technologies) and A555, K18C–A350 (1.44 ± 0.36 nM) and K18C–A555 (2.47 ± 0.05 nM), respectively . Therefore, K18C/D140H–A555 exhibited a more potent affinity for heme with no heme degrading ability and serves as a better heme sensor than those previously reported. To evaluate the practicability of heme sensors for biological samples, the rat hepatic–microsomal fraction was subjected to the heme quantification with K18C/D140H–A555. The sample was prepared as reported previously . Microsomal protein was determined by bicinchoninic acid protein assay. Here, 2 ml of 100 nM K18C/D140H–A555 in 20 mM potassium phosphate buffer (pH 7.4) was titrated with the microsomal sample (standard curve of the experiment is shown in of the supplementary material). To quantify the total heme (i.e., protein-bound heme plus free heme), the sample was treated with acidic acetone, which is a common method for extracting heme from hemoproteins . The spectral changes in K18C/D140H–A555 during the titration with the undenatured and acidic acetone-treated samples are shown in B. The titration with the intact sample exhibited gradual but modest quenching of the fluorescence emission, whereas the denatured sample induced significant quenching. These experiments were performed with six individual samples, and heme concentration was calculated as nanomoles per milligram of protein. The 0.019 ± 0.003 and 1.757 ± 0.163 nmol heme/mg protein were quantified for the undenatured and acidic acetone-treated samples, respectively. Fluorescent measurements with wavelength in the visible region seem to be desirable for the heme sensor when it is applied for biological samples. Indeed, we failed determination of free heme in rat liver homogenate by using K18C–A350 previously reported , which has excitation and emission at 353 and 430 nm, respectively, as well as K18C/D140H–A350, K18C/D140H labeled with A350, due to the background emissions in the near-UV region (see in supplementary material). The results obtained with K18C/D140H–A555 demonstrated that the hepatic microsomal fraction contained free and total heme in a ratio of nearly 1:100. To our knowledge, this is the first example of simultaneous determination of free heme and protein-bound heme in rat hepatic microsomal fraction. It is noteworthy that free heme exists in the sample at such a small population compared with total heme, although the concentration of free heme obtained here might be underestimated possibly due to an undesirable matrix effect in the sample such as a nonspecific heme binding. It is also noted that the concentration of total heme is 2-fold larger than that measured previously by the pyridine hemochromogen method (∼0.9 nmol/mg protein) , suggesting that thorough extract of heme with acidic acetone might be important for determination of the concentration of total heme.