Secondary structure content of proteins in
molten globule state is relatively constant while the
quantity of tertiary structures clearly declines due to
alterations in side-chain packing. In the present study, we
analyze the MG state of lipase-3646 for the first time. We
introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state
as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was
found that at pH 3.0 the protein acquires a MG state.
Applying far-UV circular dichroism (CD), our analysis on
the secondary structure of lipase-3646 revealed a slight
change in the MG state intermediate (pH 3.0) compared to
the native state (pH 8.5), which this amount of change is
common for MG. Measurements in near-UV CD also
showed a significant change in the enzyme conformation at
pH 3.0 in comparison with the pH 8.5 wherein the protein
acquires its native structure. Quenching the fluorescence by
applying acrylamide, the amount 23 and 35 M-1 were
measured at pHs 8.5 and 3.0 respectively for stern–volmer
constant (KSV). An increase in the enzyme molecular
volume in the MG state was confirmed by gel filtration
chromatography.