Angelw: three channel imaging reveals the regulation of gas transmitters on phosphatase activity in living cells

Enzyme is an important living substance, which participates in almost all biological reactions in vivo, such as protein synthesis, energy production and utilization, and gene expression in cells Therefore, the level and activity of enzymes significantly affect the physiological functions of organisms, and are closely related to various human diseases Phosphatase is an important member of hydrolase family, which regulates their biological functions by dephosphorizing proteins or other enzymes The activity of phosphatase itself is controlled by other upstream signal molecules such as ROS and gas transmitters Among these signal molecules, hydrogen sulfide (H 2S) is an important regulator Therefore, it is very important to explore the relationship between H 2S level and phosphatase activity in living cells Recently, Professor Zhang Zhongping's research group of Anhui University School of chemistry and Chemical Engineering designed and synthesized an integrated recognition probe n3-cr-po4 based on H 2S and phosphatase sensitive fluorophores, which can detect H 2S level and phosphatase activity in living cells at the same time Relevant achievements were published on angel Chem Int ed (DOI: 10.1002 / anie 201811391) under the title of "revealinggasotransmitter regulation to photosphatase activity in live cells by three channel imaging correlation" The corresponding authors are Professor Zhang Zhongping and Dr Zhang Ruilong In this paper, coumarin (emission wavelength is 445 nm) and rhodol (emission wavelength is 545 nm) are selected to construct the probe The main considerations are: 1 The distance between the emission peaks of the two can reach 100 nm, which effectively avoids the spectral overlap; 2 The emission spectrum of coumarin overlaps with the absorption spectrum of rhodol, which has the condition of fluorescence energy resonance transfer (FRET) The probe n3-cr-po 4 was composed of n 3-Modified coumarin and phosphoric acid modified rhodol by piperazine N 3 has a strong electron absorption effect and inhibits the blue fluorescence of coumarin, while phosphoric acid inhibits the green fluorescence of rhodol On the one hand, in the presence of H 2S, N 3 is reduced to NH 2 and the probe emits blue fluorescence; on the other hand, in the presence of phosphatase, the phosphate group is cut off and the probe emits green fluorescence; while in the coexistence of H 2S and phosphatase, due to fret Green fluorescence can only be detected at the excitation wavelength of coumarin, and the signal is weaker than that induced by a single phosphatase (Fig 1a) (source: angelw Chem Int ed.) obtained the probe n3-cr-po 4, and the author explored its response to H 2S and alkaline phosphatase (ALP) Firstly, the response of the probe to H 2S is studied In Tris HCl buffer, there are two weak emission peaks at 445 and 545 nm under 360 nm excitation With the addition of H 2S, the emission peak of the probe increases at 445 nm (Fig 1b) When the concentration of H 2S was 70 μ m, the fluorescence intensity reached the maximum value (increased by 13 times) Even under the condition of 1 NMH 2S, the fluorescence intensity of the probe is still enhanced about 1.4 times, which indicates that the probe is highly sensitive to H 2S In addition, the weak emission peak at 545 nm did not change with the addition of H 2S Then the response of the probe to ALP was studied With the addition of ALP, the fluorescence intensity of the probe increased significantly at 545 nm under 510 nm excitation (Fig 1c) When ALP concentration was 2 U / L, the fluorescence intensity increased 23 times However, under the condition of 0.01 U / L ALP, the fluorescence intensity increased by 2 times, indicating that the probe was highly sensitive to phosphatase Then, the response of the probe in the presence of H 2S and ALP was investigated Under 360 nm excitation, the mixed solution of 10 μ m probe and 50 μ M H 2S showed blue fluorescence emission at 445 nm When ALP was added into the solution, the fluorescence intensity of the emission peak at 445 nm decreased rapidly, while that at 545 nm increased about 7 times (Fig 1D) These phenomena confirmed the occurrence of FRET In addition, the author carried out a probe selectivity experiment and found that the common molecules (amino acids, peptides, proteins, enzymes and inorganic salts, etc.) in the cell had no significant effect on the fluorescence of the probe (Fig 1E) Subsequently, the authors treated HeLa cells with a 10 μ m probe and found that channels ch1 (detection of H 2S), CH2 (detection of phosphatase) and CH3 (detection of FRET correlation) showed strong fluorescence signals (Fig 2a) In order to further study the cause of fluorescence, a negative control experiment was carried out with phosphatase inhibitor (Na 3VO 4) and H 2S producing enzyme inhibitor (AOAA) When Na 3VO 4 and the probe were incubated with the cells in turn, the fluorescence signal of CH 2 and CH 3 decreased significantly, while the fluorescence signal of CH 1 increased The authors speculate that the inhibition of phosphatase activity may cause the interruption of FRET In another control group, after AOAA was added, the fluorescence signal of ch1 weakened, while the fluorescence signal of CH2 and CH3 became extremely weak, which was roughly equivalent to the fluorescence signal intensity when Na 3VO 4 was added (Fig 2b) This suggests that the decrease of H 2S level may lead to the inactivation of phosphatase in living cells (source: angel Chem Int ed.) in order to verify the effect of H 2S on phosphatase activity, the author pre treated the cells with H 2S scavenger PMA to reduce the level of H 2S in the cells, and then added 10 μ m probe for CO incubation With the increase of PMA dose, the fluorescence signals of the three channels gradually weakened, and the fluorescence signals in CH2 and CH3 almost disappeared (Fig 3a and b) This indicated that the decrease of H 2S level greatly inhibited the activity of phosphatase, and confirmed that H 2S as an activator could maintain the normal activity of phosphatase in cells The authors further treated HeLa cells with different doses of H 2S, and then incubated them with 10 μ m probe It was found that with the increase of H 2S, the fluorescence signal of CH 1 increased gradually, while that of CH 2 and CH 3 decreased gradually (Fig 3C and D) The results showed that when the level of H 2S deviated from the normal level, the activity of phosphatase decreased significantly (source: angelw Chem Int ed.) in a word, the author designed and synthesized a double response fluorescence probe n3-cr-po4, which produces two distinguishable fluorescence signals under the action of H2S and phosphatase Combined with the effect of fret, three channel imaging was established to detect the level of H 2S and phosphatase activity in living cells, and to clarify the correlation between phosphatase activity and H 2S level The probe has important reference value in exploring the regulation mechanism of enzyme activity.