1. Synchronous map Asynchronous map Order 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 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. STST time Experiment 1: 30 mg mL -1 BSA 0.5 mg mL -1 Proteinase K BSA structure Experimental Data matrix C 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 Retrieved conc. profiles Results not in agreement with 2D-CoS. Additional processes ignored? Retrieved spectra 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 CaF 2 -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 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 Experiment 1 matrix Experiment 2 matrix Experiment 3 matrix S C C C Experiment 1 residuals Experiment 2 residuals Experiment 3 residuals 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 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 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) Asynchronous map 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.