Study of Conformational Stability of DNase from Hepatopancreas of the Crab Paralithodes camtschatica

T. I. Vakorina,^1,2 N. I. Menzorova,¹ and V. A. Rasskazov¹

¹Pacific Institute of Bioorganic Chemistry, Far East Branch of Russian Academy of Sciences, pr. 100-letiya Vladivostoka 159, Vladivostok-22, 690022 Russia; fax: (7-4232) 31-4050; E-mail: rasskas@piboc.marine.su

²To whom correspondence should be addressed.

Submitted May 14, 1997; revision submitted September 12, 1997.

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UV and CD spectra of DNase K from crab hepatopancreas were recorded. The number of tyrosine and tryptophan residues in the enzyme was calculated using the second derivative of the UV spectrum. The percentage of canonical secondary structures was calculated using the CD spectrum. The tertiary structure of the enzyme is well developed and its secondary structure is highly ordered and predominantly composed of antiparallel folded beta-sheets. Thermal denaturation was studied by CD spectroscopy. Crab pancreas DNase K irreversibly denatures at 67°C. This process is cooperative and includes two stages according to the rapid change in the CD spectrum at 70°C and the position of an isodichroic point (210 nm in all spectra at 20-90°C). Acid-base titration of the enzyme results in partially reversible conformational changes at pH below 4.0 and above 10.0. This is associated with decrease in beta-sheets and increase in beta-coils and unordered structures in the secondary structure of DNase K.

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KEY WORDS: DNase K, secondary and tertiary structure, UV spectroscopy, circular dichroism, isodichroic point.

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