image: The illustration is a summary map of the organellar pH measured with a single pH-sensitive probe, SITE-pHorin, which possesses an ultra-pH sensitivity enhanced by the quantum entanglement effect. As illustrated, the nucleus, the endoplasmic reticulum, the Golgi substacks, the peroxisome, the lysosome, the endosomes, and the mitochondrial subcompartments were labeled with the peaks and standard deviations of their pH measurements. Pseudocolors were used to match the intracellular pH gradients.
Credit: ©Science China Press
Precise control of pH is essential for optimal metabolism and overall cellular function. Human blood pH is strictly maintained between 7.35 and 7.45; any deviation triggers acidemia or alkalemia and precipitates multiple diseases. Aberrant organellar pH is likewise pathogenic: mitochondrial acid–base imbalances are tightly linked to cancer and neurodegeneration, Golgi pH shifts disturb glycosylation to promote cancer and cutis laxa, and lysosomal pH dysregulation cripples the degradative system, fueling neurodegeneration, inflammation, autoimmunity, and metabolic disorders. Accurate measurement and real-time monitoring of organellar pH not only deepen our understanding of complex disease mechanisms but also provide invaluable clues for diagnosis and therapeutic intervention.
Recently, Prof. Jian-Sheng Kang’s team at the First Affiliated Hospital of Zhengzhou University published a research article entitled “A unified intracellular pH landscape with SITE-pHorin: a quantum-entanglement-enhanced pH probe” in Science China Life Sciences. The team developed a pH-sensitive fluorescent probe endowed with quantum-entanglement characteristics that exhibits single-excitation/dual-emission and covers an unusually broad response range of pH 3.5-9.0, enabling accurate pH mapping of the cytosol, nucleus and individual organelles.
From mTurquoise2 to SITE-pHorin
Starting from the brightest monomeric fluorescent protein mTurquoise2 (quantum yield 93 %), the team first introduced mutations S65T and W66Y into its chromophore. Although these two substitutions rendered the protein pH-sensitive, single-excitation/dual-emission behaviour was absent. Crystal-structure analysis revealed that mTurquoise2 adopts an 11-stranded β-barrel and that the W66Y substitution repositioned Asp148. Saturated mutagenesis at Asp148 was therefore performed, followed by screening for pH-responsive variants. Additional mutations C48S and T203C were introduced to optimize folding and brightness. The final quintuple mutant (C48S/S65T/W66Y/D148G/T203C) displayed a wide pH response (3.5-9.0), high quantum yield, and the desired single-excitation/dual-emission property that allows ratiometric quantification. The probe dubbed SITE-pHorin (Single-excitation Two-Emission probe for intracellular SITEs) offers a spatiotemporally resolved tool for precise organellar pH imaging in living cells.
The quantum entanglement mechanism of the pH ultrasensitivity of SITE-pHorin
Then, they compared the crystallographic structure of SITE-pHorin at different pH values, and combined with charge-state analysis, theoretical modelling, and quantum-chemical calculations, uncovered a quantum-entanglement interaction between the chromophoric phenol and the phenolic side chain of Tyr182 within the barrel. They found that this non-covalent coupling boosts the probe’s sensitivity to at least twice that of the best conventional sensors, representing a leap in pH-detection performance.
SITE-pHorin targeted to organelles and their subcompartments
To evaluate the probe’s performance in living cells, the Kang team fused the SITE-pHorin to well-validated organelle-targeting peptides or proteins, the researchers systematically optimized localization signals, and accurately measured the pH of individual compartments. Most notably, they resolved the long-standing controversy over mitochondrial pH: in COS-7 cells, the cristae were pH 6.60 ± 0.40, the intermembrane space 6.95 ± 0.30, and the matrix exhibited two subpopulations at 7.20 ± 0.27 and 7.50 ± 0.16. Thus, the true ΔpH from cristae to matrix reaches 0.6-0.9 units, substantially exceeding the canonical 0.4-0.6, indicating that previous estimates of proton-motive force were underestimated and providing solid data for re-evaluating cellular energetics and related pathological mechanisms. Since most fluorescent proteins are inactivated in acidic environments, accurate lysosomal pH has been elusive; utilizing SITE-pHorin, the lysosomal lumen was determined as pH 4.79 ± 0.17, perfectly matching the optimal working range of pH 4.5-5.0. In the end, the Kang team successfully constructed a comprehensive intracellular pH map (see figure).
Li, S.A., Meng, X.Y., Zhang, S., Zhang, Y.J., Yang, R.Z., Wang, D.D., Yang, Y., Liu, P.P., and Kang, J.S. A unified intracellular pH landscape with SITE-pHorin: a quantum-entanglement-enhanced pH probe. Sci China Life Sci. doi: 10.1007/s11427-025-2971-5
Journal
Science China Life Sciences
Method of Research
Experimental study