Two-photon excitation (2PE) of "caged" biomolecules represents a powerful method to investigate the temporal and spatial relevance of physiological function in real time and on living tissue, because the excitation volume can be restricted to 1 fL. Additionally, low-energy IR light is used, which minimizes tissue destruction and enables deeper penetration into tissue preparations. Exploitation of this technology for studying cell physiology requires the further development of photoremovable protecting groups with sufficient sensitivity to 2PE for use in "caged" compounds. 8-Bromo-7-hydroxyquinoline (BHQ) is efficiently photolyzed by classic 1PE (365 nm) and 2PE (740 nm) under simulated physiological conditions (aqueous buffer of high ionic strength, pH 7.2) to release carboxylates, phosphates, and diols-functional groups commonly found on bioactive molecules such as neurotransmitters, nucleic acids, and drugs. It is stable in the dark, soluble in water, and exhibits low levels of fluorescence, which will enable use in conjunction with fluorescent indicators of biological function. BHQ-protected effectors are synthetically accessible. Stern-Volmer quenching, time-resolved infrared (TRIR), and (18)O-labeling experiments suggest that the photolysis occurs through a solvent-assisted photoheterolysis (S(N)1) reaction mechanism on the sub-microsecond time scale. BHQ has the requisite photochemical and photophysical properties as a photoremovable protecting group to regulate the action of biological effectors in cell and tissue culture with light, especially 2PE.

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca(2+) by the cardiac SR Ca(2+) pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphorylation) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys --> Ala mutant). The results show that activation of SERCA2a dephosphorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca(2+) pump.