Purification, characterization, and unfolding studies of arginine kinase from Antarctic krill.

The regulation of enzymatic activity and unfolding studies of arginine kinase (AK) from various invertebrates have been the focus of investigation. To gain insight into the structural and folding mechanisms of AK from Euphausia superba (ESAK), we purified ESAK from muscle properly. The enzyme behaved as a monomeric protein with a molecular mass of about 40kDa and had pH and temperature optima of 8.0 and 30°C, respectively. The Km(Arg) and Km(ATP) for the synthesis of phosphoarginine were 0.30 and 0.47mM, respectively, and kcat/Km(Arg) was 282.7s(-1)/mM. A study of the inhibition kinetics of structural unfolding in the denaturant sodium dodecyl sulfate (SDS) was conducted. The results showed that ESAK was almost completely inactivated by 1.0mM SDS. The kinetics analyzed via time-interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to biphase as SDS concentrations increased. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence showed that SDS concentrations lower than 5mM did not induce conspicuous changes in tertiary structures, while higher concentrations of SDS exposed hydrophobic surfaces and induced conformational changes. These results confirmed that the active region of AK is more flexible than the overall enzyme molecule.
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