Reversibility of Unfolding of Walleye Pollack Light Meromyosin by Heat-Treatment

Abstract

Reversibility of unfolding of α-helix in walleye pollack light meromyosin (LMM) by heat-treatment was investigated by circular dichroism spectrometry and α-chymotryptic digestion. By increasing the temperature from 5 to 50°C, α-helix content of the LMM was decreased from 88% to 15% with a major and a minor transition at around 28°C and 40°C, respectively. On the other hand, α-helix content of the LMM was increased from 15% to 68% by decreasing the temperature from 50°C to 5°C, while it was not fully recovered to the original value, suggesting that some α-helical portions were irreversibly unfolded by heating. Therefore, the LMM was heated at 50°C for 5 min, cooled to 0°C, and then digested with α-chymotrypsin to assign the reversible and irreversible unfolding regions. As a result, the 42-kDa fragment and some smaller peptides which were hardly formed by the digestion of unheated LMM were newly formed. The 42-kDa fragment was identified as an N-terminal 2/3 region of the LMM by the amino acid sequencing of both N- and C-terminal regions, while most of the smaller peptides were derived from the C-terminal 1/3 region of the LMM. The 42-kDa fragment showed 90% α-helix content and its unfolding upon heating was almost reversible unlike the original LMM. From these results, we propose that the N-terminal 2/3 and C-terminal 1/3 regions of the pollack LMM are reversiblv and irreversibly unfolding regions, respectively, by the heat-treatment.