In situFTIR studies on mammalian cells

Abstract

In this work, we describe the application of FTIR spectroscopy to study lipids and proteins, in cellular systems during heating and cooling. Various spectral analysis methods are described to simultaneously study membrane phase behavior and heat-induced protein denaturation in cells. Membrane phase behavior was studied by following the temperature dependence of the CH2stretching vibration bands. Protein denaturation was studied using the amide-I and -III bands. Both amide-I and amide-III band analysis show that heat-induced denaturation of proteins within the cells is typically associated with an increase in extendedβ-sheet structures and a concomitant decrease in α-helical structures.

Sperm Membrane Behaviour during Cooling and Cryopreservation

“…Absorbance bands arising from endogenous biomolecules are best visible in the dried sample, as water absorbs most of the infrared light causing overlap of bands. Characteristic bands arising from cellular proteins are the amide‐I (1600−1700 cm −1 ), amide‐II (1500−1600 cm −1 ) and amide‐III band (1330−1200 cm −1 ) (Wolkers and Hoekstra ; Wolkers and Oldenhof ). Protein denaturation is evident as changes in the shape of these bands (Wolkers and Oldenhof , ).…”

“…Characteristic bands arising from cellular proteins are the amide‐I (1600−1700 cm −1 ), amide‐II (1500−1600 cm −1 ) and amide‐III band (1330−1200 cm −1 ) (Wolkers and Hoekstra ; Wolkers and Oldenhof ). Protein denaturation is evident as changes in the shape of these bands (Wolkers and Oldenhof , ). The region below 1500 cm −1 is designated as the finger print region and contains bands that are difficult to assign to specific group vibrations.…”

“…The level of membrane conformational disorder (fluidity) is typically evaluated using the band position of the symmetric CH 2 ‐stretching vibration band at approximately 2850 cm −1 . This band shifts to lower wavenumber position upon packing of the acyl chains coinciding with a fluid‐to‐gel phase transition (Caffrey ; Mantsch and McElhaney ; Wolkers and Oldenhof , ). Figure a shows membrane phase behaviour of bull and stallion sperm at suprazero temperatures analysed using FTIR.…”

Protein secondary structure and solvent accessibility of proteins in decellularized heart valve scaffolds

“…It should be noted that the amide-I band also contains a contribution from the H 2 O scissoring band. In the region below 1500 cm −1 a variety of characteristic molecular group frequencies can be observed that are difficult to assign but the shape of the spectra in this region is characteristic and provides a 'finger print' of the tissue [14].…”

“…When proteins in tissues are analyzed by FTIR, the amide bands provide information on the overall protein secondary structure of all endogenous proteins [11]. Temperature scanning FTIR studies, in which spectra are acquired while changing the temperature of the sample, can be used to detect heat-induced protein denaturation in cells and tissues [11,14].…”