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)
BSA hydrolysis
monitored by FTIR
of infrared spectroscopy. Two Dimensional Correlation Spectroscopy (2DCoS) has been used to
as COO(1594 cm-1)
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
Wavenumber (cm-1)
spectra and concentration profiles of the different species involved in the reaction.
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
1520
1520
1540
1540
1560
1560
1580
1600
1620
1640
Experimental
Data
matrix
1680
3) 1675, 1616 cm-1 (β-turn,βsheets)
1650
1600
1550
Wavenumber (cm-1)
Conformational changes with different kinetics than the proteolysis
proccess are detected
Analysis of residuals using 2D-CoS
Synchronous
map
(a)
Wavenumber (cm-1)
Retrieved spectra
0.014
Asynchronous
map
(b)
1520
1520
1540
1540
1560
1560
1580
1600
1620
1640
1580
1600
1620
1640
1660
1660
1680
1680
1648 cm-1 (Amide I)
0.012
25
1650
1600
1550
Wavenumber (cm-1)
1650
1600
1550
Wavenumber (cm-1)
0.01
20
Absorbance
Concentration (mg/ml)
1640
1680
C
30
2) 1641, 1594 cm-1 (disordered
structures, COO-)
1620
1660
Residuals
matrix
Retrieved conc. profiles
 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
1600
1650
1600
1550
Wavenumber (cm-1)
Reaction conditions: 60°C 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 (60°C) 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.
15
1594 cm-1
( as COO-)
0.008
Two processes have been excluded from the MCR model:
 Changes in the amide I band involving -helix conformation (1654cm-1)
 Formation of β-sheet aggregates (1616 cm-1)
0.006
10
0.004
5
0
0.002
0
50
100
150
200
Time (min)
250
Residuals inspection
0
1700 1680 1660 1640 1620 1600 1580 1560 1540 1520
Wavenumber (cm-1)
300
Results not in agreement with 2D-CoS.
Additional processes ignored?
Analysis of residuals using 2D-CoS
Asynchronous
map
(b)
1520
Experiment 1: 30 mg
BSA
0.5 mg mL-1 Proteinase K
C
Experiment
2
matrix
Experiment 2: 50 mg mL-1 BSA
0.5 mg mL-1 Proteinase K
Experiment
2
residuals
C
Experiment
3
matrix
Experiment 3: 30 mg mL-1 BSA
Blank run without enzyme
Experiment
1
residuals
Experiment
3
residuals
C
 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
Exp.1
1560
1580
1600
1620
1640
1660
1680
1650
1600
1550
Wavenumber (cm-1)
Asynchronous
map
(a)
1520
1540
Wavenumber (cm-1)
S
Experiment
1
matrix
STEPS
Wavenumber (cm-1)
1540
mL-1
Exp.3
1580
1600
1620
1640
1680
Retrieved concentration profiles
50
Experiment 1
Experiment 3
20
10
native albumin
30
20
10
0.012
Absorbance
30
0.014
40
Concentration (mg/ml)
40
Concentration (mg/ml)
Concentration (mg/ml)
0.016
Experiment 2
40
1650
1600
1550
Wavenumber (cm-1)
Retrieved spectra
50
30
20
0.01
proteolysis
product
0.008
0.004
10
0
50
100
150
200
Time (min)
250
300
Fast
0
0
50
100
150
200
Time (min)
250
300
Slow
0
0
50
100
150
200
Time (min)
native albumin → unfolded albumin → proteolysis product
60º
60º, Proteinase K
250
300
Unfolding of BSA before proteolysis and appearance of -sheet
aggregates were detected.
 The combined use of MCR and 2DCoS is a powerful approach for

0.006
unfolded
albumin
0.002
0
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 βsheets 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 β-sheet aggregated
structures have not been
completely modeled in the
blank experiment.
1560
1660
50
Order of spectral changes:
1) 1654 cm-1 (-helix)
1580
1660
S
STEPS
Asynchronous
map
(b)
Wavenumber (cm-1)
Conformational changes previously reported:
-reversible in the temperature range of 42-50°C.
-irreversible unfolding of -helices in the temperature
range of 52-60°C
- unfolding progresses and -aggregation begins above
60°C
Synchronous
map
(a)
Amide I
(1651 cm-1)
Wavenumber (cm-1)
The hydrolysis of bovine serum albumin with protease K at 60 ºC has been studied by means
BSA structure
ST
0
1700 1680 1660 1640 1620 1600 1580 1560 1540 1520
Wavenumbers (cm-1)
native albumin: 1651 cm-1 (-helix )
unfolded albumin: 1648 cm-1 (disordered
strutures ) 1616 cm-1(β-sheet)
proteolysis product: 1594 cm-1 (COO-),
1616 cm-1 (β-sheet) 1670 cm-1 (β-turn)
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.