Effects of aspartic acid and potassium chloride on arginine kinase from shrimp.
The aspartic acid (Asp)-induced unfolding and the salt-induced folding of arginine kinase (AK) were studied in terms of enzyme activity, intrinsic fluorescence emission spectra, 1-anilino-8-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that Asp caused inactivation and unfolding of AK with no aggregation during AK denaturation. The unfolding of the whole molecule and the inactivation of AK in different Asp concentrations were compared. Much lower Asp concentration was required to induce inactivation than to produce significant conformational changes of the enzyme molecule. However, with further addition of Asp, the molar ellipticity at 222 and 208 nm, the wavelength shift and the emission intensity of ANS hardly changed. Asp denatured AK was reactivated by dilution. In addition, potassium chloride (KCl) induced the molten globule state with a compact structure after AK was denatured with 7.5 mM Asp. These results collectively elucidate the osmotic effect of Asp anions for the molten globule formed during unfolding process. They also suggest that the effect of Asp differed from that of other denaturants such as guanidine hydrochloride or urea during AK folding. The molten globule state indicates that intermediates exist during AK folding.
Tang
Hong-min
HM
Department of Chemistry, Guizhou University for Ethnic Minorities, Guiyang 550025, China. thm@mail.gznc.edu.cn
Yang
Yin-ye
YY
Zhang
Song-fu
SF
D000818:Animals; D001122:Arginine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000494:pharmacology; D002942:Circular Dichroism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D046912:Multiprotein Complexes; D033561:Penaeidae / Q000201:enzymology*; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D046912:Multiprotein Complexes; D001224:Aspartic Acid; D011189:Potassium Chloride; D001122:Arginine Kinase