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Wavenumber (cm-1)155016001650

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Synchronous map Asynchronous mapOrder of spectral changes:

1)1654 cm-1 (-helix)

2) 1641, 1594 cm-1

(disordered structures, COO-)

3) 1675, 1616 cm-1 (β-turn,β-sheets)

Conformational changes with different kinetics than the proteolysis proccess are detected

DETECTION OF ALBUMIN UNFOLDING PRECEDING PROTEOLYSIS

BY MEANS OF FT-IR SPECTROSCOPY USING 2D-CoS AND MCR

DETECTION OF ALBUMIN UNFOLDING PRECEDING PROTEOLYSIS

BY MEANS OF FT-IR SPECTROSCOPY USING 2D-CoS AND MCR

Institute of Chemical Technologies and Analytics, Vienna University of Technology (Austria)

Department of Physical and Analytical Chemistry, University of Jaén (Spain)

María José Ayora-Cañada, Ana Domínguez-Vidal, Bernhard Lendl

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.

ST

Experiment 1: 30 mg mL-1 BSA0.5 mg mL-1 Proteinase K

BSA structure

Experimental Data

matrixC

S

Residuals matrix

BSA hydrolysis monitored by FTIR

Amide I (1651 cm-1)

as COO-

(1594 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

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

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

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Retrieved conc. profiles

Results not in agreement with 2D-CoS. Additional processes ignored?

Retrieved spectra

15201540156015801600162016401660168017000

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1648 cm-1 (Amide I)

1594 cm-1

( as COO-)

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.

Analysis of residuals using 2D-CoS

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Asynchronous mapSynchronous map

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)

Residuals inspection

Experiment1

matrix

Experiment2

matrix

Experiment3

matrix

S

C

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Experiment1

residuals

Experiment2

residuals

Experiment3

residuals

Experiment 2: 50 mg mL-1 BSA0.5 mg mL-1 Proteinase K

Experiment 3: 30 mg mL-1 BSABlank run without enzyme

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

Analysis of residuals using 2D-CoS

Retrieved spectraRetrieved concentration profiles

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native albumin

proteolysis product

unfolded albumin

Experiment 3 Experiment 2Experiment 1

native albumin → unfolded albumin → proteolysis product 60º, Proteinase K

SlowFast

60º

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)

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Exp.1

Exp.3

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 β-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 β-sheet aggregated structures have not been completely modeled in the blank experiment.

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.

synchronous map

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