Diapositiva 1

1
DETECTION OF ALBUMIN UNFOLDING PRECEDING
PROTEOLYSIS BY MEANS OF FT-IR SPECTROSCOPY USING
2D-CoS AND MCR
María José Ayora-Cañada, Ana Domínguez-Vidal,
Bernhard Lendl
Institute of Chemical Technologies and Analytics,
Vienna University of Technology
(Austria) Department of Physical and Analytical
Chemistry, University of Jaén (Spain)
Introduction
Two dimensional correlation spectroscopy
ST
The hydrolysis of bovine serum albumin with
protease K at 60 ºC has been studied by means of
infrared spectroscopy. Two Dimensional
Correlation Spectroscopy (2DCoS) has been used to
study spectral changes in the reaction. The use
of Multivariate Curve Resolution-Alternating
Least Squares method applied to infrared
measurements allowed the recovery of pure
infrared spectra and concentration profiles of
the different species involved in the reaction.
Synchronous map
Asynchronous map

• Order of spectral changes
• 1654 cm-1 (?-helix)
• 2) 1641, 1594 cm-1 (disordered structures, COO-)
• 3) 1675, 1616 cm-1 (ß-turn,ß-sheets)

Bovine serum albumin (BSA) is a single
polypeptide chain built from 583 amino acid
residues with a molecular mass of 66500Da. The
secondary structure of BSA is composed of 67
?-helix, 10 turn and 23 extended chain and no
?-sheet is present

• Conformational changes previously reported
• reversible in the temperature range of 42-50C.
• irreversible unfolding of ?-helices in the
  temperature range of 52-60C
• unfolding progresses and ?-aggregation begins
  above 60C

Experimental
Conformational changes with different kinetics
than the proteolysis proccess are detected
Reaction conditions 60C in phosphate buffer
prepared in deuterium oxide (pD 7.4). Proteinase
K 0.5 mg ml-1 BSA 30 mg ml-1 Thermostatized
flow cell (60C) equipped with CaF2-windows (4 mm
thick) and polytetrafluoroethylene spacer (50 ?m
optical path) Bruker Equinox 55 FT-IR
spectrometer with narrow band MCT detector.
Resolution 2 cm-1, averaging 128 scans.
Background spectrum was recorded with the flow
cell filled with buffer. Infrared spectra were
recorded every 2 min during 320 min.
Multivariate curve resolution -Alternating least
squares (MCR-ALS)
STEPS ? 2 components explained 99.99 of
variance ? Evolving factor analysis (EFA) was
used to build initial estimates of concentration
profiles ? Optimization by alternating least
squares. Constrains nonnegativity (spectra and
conc. profiles, unimodality (conc. profiles),
closure
Residuals inspection
Analysis of residuals using 2D-CoS
MCR-ALS with matrix augmentation
The presence of a band at 1651 cm-1 due to the
native albumin can be justified because the
denaturation is so fast that it is very difficult
to model. Spectral contributions of ß-sheets
structures are of minor importance in the
experiments involving the enzyme probably because
formation of these structures is disabled by the
proteolysis process The heat-induced
conformational changes producing ß-sheet
aggregated structures have not been completely
modeled in the blank experiment.
time
STEPS ? 3 components explained 99.85 of
variance ? Evolving factor analysis (EFA) to
build initial estimates of concentration
profiles ? Optimization by alternating least
squares. Constrains nonnegativity (spectra and
conc. profiles, unimodality (conc. profiles),
closure
Experiment 1 30 mg mL-1 BSA 0.5 mg mL-1
Proteinase K
Experiment 2 50 mg mL-1 BSA 0.5 mg mL-1
Proteinase K
Experiment 3 30 mg mL-1 BSA Blank run
without enzyme
Retrieved spectra
Retrieved concentration profiles
Conclusions
? Unfolding of BSA before proteolysis and
appearance of ?-sheet aggregates were detected. ?
The combined use of MCR and 2DCoS is a powerful
approach for the study of protein reactions using
FT-IR ? 2DCoS applied to the residuals from MCR
is useful to get more information about the
modeling process.
native albumin 1651 cm-1 (?-helix ) unfolded
albumin 1648 cm-1 (disordered strutures ) 1616
cm-1(ß-sheet) proteolysis product 1594 cm-1
(COO-), 1616 cm-1 (ß-sheet) 1670 cm-1 (ß-turn)