alpha-chymotrypsin and sodium-perchlorate

RecA is a naturally aggregating Escherichia coli protein that catalyzes the strand exchange reaction utilized in DNA repair. Previous studies have shown that the presence of salts influence RecA activity, aggregation, and stability and that salts stabilize RecA in an inverse-anionic Hofmeister series. Here we utilized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and circular dichroism (CD) to investigate how various Hofmeister salts alter the water structure and RecA solvation and aggregation. Spectroscopic studies performed in water and deuterium oxide suggest that salts alter water O-(1)H and O-(2)H stretch and bend vibrations as well as protein amide I (or I') and amide II (or II') vibrations. Anions have a much larger influence on water vibrations than cations. Water studies also show increased water-water and/or water-ion interactions in the presence of strongly hydrated SO4(2-) salts and evidence for decreased interactions with weakly hydrated Cl(-) and ClO4(-) salts. Salt-water difference infrared spectra show that kosmotropic salts are more hydrated than chaotropic salts. Interestingly, this is the opposite trend to the changes in protein solvation. Infrared spectra of RecA show that vibrations associated with protein desolvation were observed in the presence of SO4(2-) salts. Conversely, vibrations associated with protein solvation were observed in the presence of Cl(-) and ClO4(-) salts. Difference infrared studies on the dehydration of model proteins aided in identifying changes in RecA-solvent interactions. This study provides evidence that salt-induced changes in water vibrations correlate to changes in protein solvent interactions and thermal stability.

Topics: Acrylic Resins; Chlorides; Chymotrypsin; Circular Dichroism; Escherichia coli Proteins; Ions; Myoglobin; Perchlorates; Protein Unfolding; Rec A Recombinases; Sodium Compounds; Spectroscopy, Fourier Transform Infrared; Sulfates; Water

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.

Topics: 2-Propanol; Animals; Catechol Oxidase; Catechols; Chymotrypsin; Dopamine; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Hemocyanins; Hydrogen-Ion Concentration; Kinetics; Levodopa; Molecular Weight; Monophenol Monooxygenase; Palinuridae; Perchlorates; Sodium Compounds; Sodium Dodecyl Sulfate; Substrate Specificity; Trypsin