Hirsch R E, Zukin R S, Nagel R L
Biochem Biophys Res Commun. 1986 Jul 16;138(1):489-95. doi: 10.1016/0006-291x(86)90307-4.
In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.
过去，由于使用直角光学器件无法检测到与血红素蛋白结合的外源性荧光团的荧光，所以只有在去除血红素后才会研究其荧光发射。使用前表面荧光测定法，可以检测到来自与血红蛋白结合的探针的显著稳态发射信号。这是关于检测与血红素蛋白结合的探针的外源性荧光的首次报告。我们还证明，外源性探针5-碘乙酰氨基荧光素与血红蛋白共价结合，具体结合位点为β93位半胱氨酸。如血红蛋白晶体学研究所预测的那样，配体结合导致荧光团荧光强度发生变化。进行了能量转移效率的测量。
