Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.
Papain is a proteolytic enzyme of great commercial value. It is a cysteine
protease highly expressed in Carica papaya fruit latex, but also present in
papaya leaves. Purification procedures mostly deal with the latex and
include a combination of precipitation and/or chromatographic techniques.
Due to its solubility, structure and activity characteristics, the pH and
salt content play significant roles in handling papain extracts. Here we
report a simple, rapid and easily scalable procedure for papain purification
from papaya leaves, which contain different contaminants as compared to
papaya latex. Sodium chloride precipitation of contaminants at pH 5 followed
by ammonium sulphate precipitation resulted in the removal of other leaf
proteins and protein fragments from papain solution and about a 3-fold
purification. The procedure also benefits from the suppression of
autoproteolysis and preservation of the native structure, as confirmed by
FTIR analysis, and the high recovery of activity of over 80%.
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