Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.
Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.
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游离和蛋白质结合荧光素染料的荧光特性。
DOI：
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发表时间：
1978
影响因子：
3.2
通讯作者：
G. Wick
中科院分区：
文献类型：
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作者：
K. Schauenstein;E. Schauenstein;G. Wick
用宏观荧光法研究了荧光素衍生物的激发和发射性质。用由荧光素二乙酸酯（FDA）、荧光素异硫氰酸酯（FITC）和与兔γ-球蛋白结合的FITC制备的各种浓度（0.07-100微克/ml）的游离荧光素钠溶液进行测量。记录激发和发射光谱以及恒定激发/发射波长（496/515 nm）下的荧光强度。结果表明：（1）与相同浓度的FITC相比，FDA产生的荧光强度约为两倍。(2)FITC的荧光特性在激发蓝光（λ = 496 nm）时，仅轻微改变的共轭兔γ-球蛋白。(3)然而，在UV激发（λ = 340 nm）时，可以显示出由于缀合而发生的相当大的淬灭。(4)由可见蓝光（496 nm）激发的荧光发射在0.07-2.5微克/ml的范围内随染料浓度线性增加。从5微克/毫升（10-（5）M/1）开始，所有三种化合物显示荧光强度随着浓度的进一步增加而急剧下降。这些数据的免疫荧光方法的实际方面进行了讨论。
Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.