A new fluorescence biosensor for nitric oxide detection based on cytochrome P450 55B1

Nitric oxide (NO) is an important biological signaling molecule, which plays an important role in the regulation of cell function of the nervous, vascular and immune systems. Nitric oxide also is involved in the parthenogenesis of Parkinson’s disease and tumor angiogenesis. Thus, the detection of nitric oxide is essential to understand its physiological and pathological process. Cytochrome P450 55B1 from Chlamydomonas reinhardtii is known to function as nitric oxide reductase (NOR). Here we describe the development of a new fluorescence biosensor for NO based on the NOR activity of P450 55B1. In this paper, we constructed pET28a-55b1 vector and expressed P450 55B1 in Escherichia coli successfully. We studied the interaction between P450 55B1 and NO by spectrometric method. The result show that the fluorescence intensity of iron porphyrin of P450 55B1 increase gradually with the addition of NO. When the concentration of NO is below 22.5μM, the fluorescence intensity increase linearly with the concentration of NO from a baseline value of ΔF=0.0675+0.15 CNO (in μM), which is slightly affected by potentially interfering chemicals. NO release from the rat liver homogenate stimulated by l-arginine was determined by the fluorescence NO biosensor. The results demonstrated that our strategy can be used to detect the concentration of NO in biologically samples.