Abstract
p53 over expression in yeast results in cell death with typical markers of apoptosis such as DNA fragmentation and phosphatidylserine externalization. We aimed to substitute/supplement classical fluorescent techniques (TUNEL, Annexin V, ROS detection) usually used to detect biochemical changes occurring during yeast apoptosis mediated by p53 over expression and the effect of anti-apoptotic purified molecules from Nigel (Nigella sativa) extracts on these same yeasts by the label free technique of FTIR spectroscopy. The comparison of the entire IR spectra highlighted clear modifications between apoptotic p53-expressing yeasts and normal ones. More precisely, DNA damage was detected by the decrease of band intensities at 1079 and 1048 cm-1. While phosphatidylserine exposure was followed by the increase of νsCH2 and νasCH2 bands of unsaturated fatty acids that were exhibited at 2855 and 2926 cm-1, and the appearance of the C = O ester functional group band at 1740 cm-1. In a second step, this FTIR approach was used to estimate the effect of a purified fraction of the Nigel extract. The modulation of band intensities specific to DNA and membrane status was in agreement with apoptosis supression in presence of the Nigel extracts. FTIR spectroscopy is thus proven to be a very reliable technique to monitor the apoptotic cell death in yeast and to be used as a means of evaluating the biomolecules effect on yeast survival.
Highlights
Fourier-transform infrared (FTIR) spectroscopy is a vibrational spectroscopic technique used to solve the chemical composition information of a sample and to provide its “molecular fingerprint” [1]
These findings prove that the FTIR technique is a good tool to study the biochemical changes in yeast during apoptosis
Conformational changes in 3000–2820cm−1 are reported in ethanol-stressed yeast cells [24] and can possibly be associated to biochemical changes resulting from the disorganization of the cell plasma membrane itself due to the increase of unsaturated fatty acids leading to an increase of the yeast membrane fluidity [63]
Summary
Fourier-transform infrared (FTIR) spectroscopy is a vibrational spectroscopic technique used to solve the chemical composition information of a sample and to provide its “molecular fingerprint” [1]. This technique has been previously explored for different measurement modes in many biological systems [2,3] to become a powerful tool for the cell components analysis, such as nucleic acids [4] proteins [5] and membranes [6]. Bellisola and Sorio [27] consider that FTIR spectroscopy in medical biology is an interesting emerging opportunity
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