7.2.1. Redox-Sensitive Green Fluorescent Protein (roGFP)

Formulation of redox-sensitive green fluorescent protein (roGFP) involves addition of redox reactive cysteine in GFP beta-strand 7 and 10 at site Q204 and S147. Under a reduced environment, it causes formation of disulfide linkage between two domains, which reacts to redox changes in intra/extra-cellular systems, leads to reversible ratiometric change in the intensity of fluorescence [211]. Based on roGFP, numerous probes are developed.

roGFP 1 and 2 are the first two analogues of roGFP that are differentiated based on the amino acid (T65S) unit. These may indicate conversion of dithiol/disulfide that is stimulated through ROS accumulation. These are used to examine variation in thiol/disulfide equilibrium [149]. The cysteine pair of roGFP 1 and 2 is protonated under physiological pH. Rather than direct ROS measurement, roGFP1 and 2 determine dynamic redox change. As roGFP1 and 2 undergo complete oxidation by oxidating organelles (lysosomes and endosomes), they can be appropriate for reduced environments (mitochondria, cytoplasm, peroxisomes, and nucleus) [212]. The sensitivity of roGFP also depends upon pH and speed of reaction. As intensity of roGFP fluorescence does not change quickly with change in redox condition, this indicates suitability of roGFP in monitoring the long-term redox shifts [210]. Moreover, in comparison to roGFP1, roGFP2 is easily influenced by variation in pH (ranged between 6 to 8), hence detection condition is an important aspect to consider.

Depending upon roGFP1 usage in oxidizing environment, the roGFP1-iX was formulated. The formulation of roGFP1-iX involves incorporation of the amino acids next to cys147 and mutation of H148S in roGFP1 beta-strand 7. The roGFP1-iX offers fast reaction speed and low pH sensitivity (ranged from 6 to 8) in comparison to roGFP1. This results in roGFP1-iX suitability in monitoring redox in oxidating organelles such as endoplasmic reticulum (ER) [213,214].

roGFP1 probe was modified to roGFP1-iL for sensing the redox of ER. The roGFP1-iL disulfide bonds are partly oxidized, which generates high reduction potential to examine the variation in redox condition [215]. roGFP1-iL and Grx1 in combination exhibit high sensitivity towards 2GSH or GSSG. In comparison to roGFP1, roGFP1-RX exhibits high reaction speed and dynamic range through addition of three amino acids (carrying positive charge) next to cysteine [216].

Just like roGFP1, derivatives were also created for roGFP2. The broad midpoint potential of Grx1-roGFP2-iL makes it suitable for determination of redox in ER and cytosol. In comparison to roGFP2, the derivative Grx1-roGFP2-iL exhibits higher specificity in measurement of redox potential of GSH [192]. The derivative probe roGFP2-Orp1 is H₂O₂-specific. roGFP2-Orp1 was created by combining roGFP2 with yeast peroxidase Orp1, and redox relay equivalent between the two allows the roGFP2-Orp1 probe to efficiently indicate the H₂O₂ level [217]. The pH stability of this derivative probe makes it applicable for sensing alteration in H₂O₂ levels in mitochondria and cytosol in micromole [218]. The roGFP2-Orp1 probe exhibits superior selectivity towards H₂O₂. It is important to note that none of the studies reported roGFP probes-based analysis to determine the concentration or net formation of H₂O_2. In fact, roGFP probes can only sense the variation in H₂O₂ or redox level instigated by external influences.