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Background: The pH of a biological system is a crucial determinant of the structures and reactivity of its components and cellular homeostasis of H(+) is critical for cell viability. Control and monitoring of cellular acidity are highly desirable for the purpose of studying biochemical processes in vivo., Methods: The effect of photolysis of a caged strong acid, the ester 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) is used to cause a controlled drop in pH in single cells. An isolated cell is selected under the IR microscope, irradiated with near-UV light and monitored by FTIR., Results: We demonstrate the use of FTIR spectromicroscopy to monitor light-induced acidification of the cellular medium by measuring the increased concentration of CO2 and corresponding decrease of HCO3(-) in the cell and in the surrounding medium., Conclusions: We have demonstrated a method to control and accurately monitor the changes in pH of a cellular system by coupling a caged proton-releasing agent with FTIR spectromicroscopy detection. The overall implementation of photolysis and spectroscopic detection in a microscope optical configuration ensures single cell selectivity in both acidification and monitoring. We show the viability of monitoring of pH changes by FTIR spectromicroscopy with sensitivity comparable to that of glass electrodes, better than the existing methods for determining cell pH., General Significance: Reporting the effect of small variations of cellular acidity provides a major improvement in the understanding of the interplay between molecular properties as assessed in vitro and cell physiology., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The phase behavior of binary mixtures of egg sphingomyelin and cholesterol has been inspected by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in the amide I' band region of the spectrum. Because cholesterol does not have any major absorption bands in this region, effects seen in the spectra of mixtures of sphingomyelin and cholesterol can be attributed to the change in the lipid phase and to the interaction with cholesterol. It is shown that the temperature dependence of the overall bandwidth of the amide I' band displays a phase-specific behavior. In addition, it is observed that the amide I' band for a sample exhibiting phase coexistence can be described by a linear combination of the spectra of the individual lipid phases. Description of changes in the amide I' band shape and by that the study of possible hydrogen bonding interactions of sphingomyelin with cholesterol was assisted by the use of curve fitting. It turns out that the presence of hydrogen bonding between hydroxyl group of cholesterol and carbonyl group of sphingomyelin is obscured by the complexity of different possible hydrogen bonding and coupling between the N-H (N-D) and the CO group vibrations.

The reaction of mammalian cytochrome P-450 2B4 with nitrogen monoxide and oxygen has been studied by surface-enhanced resonance Raman scattering (SERRS) to obtain sharp and definitive information in situ on the nature of the changes in the active site pocket. The initial reaction produces a six co-ordinate low spin haem-nitrogen monoxide adduct. A slower reaction leads to the irreversible formation of a five co-ordinate high-spin iron (III) haem with no nitrogen monoxide bound to it and to the nitration of an aromatic side chain, probably a tyrosine, in the proximity of the active site. In the presence of excess nitrogen monoxide, the second reaction is controlled by oxygen concentration. The sequence of events corresponds to the biphasic inhibition induced by NO in other cytochromes P-450 and peroxidases and is postulated to occur by the formation of a nitrating agent at the haem followed by diffusion to the tyrosine. The nitrated amino acid and the oxidation and spin state of the haem are observed easily by SERRS with low concentrations of protein making it a particularly suitable method for the investigation of reactions of NO in complex biological matrices.