Differential scanning calorimetry (DSC) was employed for studying the thermal unfolding of myosin and its rod part prepared from carp acclimated to 10 and 30 degrees C. Differences in the thermal stability reflecting structural properties were clearly demonstrated by the DSC data obtained at pH 8.0 in 0.6 M KCl for the two types of carp myosin and rod. The transition temperatures on myosin and rod given by the major peaks for the 10 degrees C-acclimated carp were 33.9 and 47.4 degrees C and 33.0 and 44.0 degrees C, respectively, assuming two endotherms for this type. Since the shape of the first peaks at 33.9 and 33.0 degrees C was not symmetrical, two peaks having similar transition temperatures overlapped in this temperature range. When the data were analyzed using three endotherms, the three transition temperatures obtained for myosin and rod were 32.8, 34.9, and 47.4 degrees C and 32.9, 33.4, and 44.1 degrees C, respectively. Thus, the position of the first peak for the 10 degrees C-acclimated carp myosin did not change even after removal of the large subfragment-1 part, but the transition of the second peak shifted to a lower temperature by about 3 degrees C. The myosin and rod from carp acclimated to 30 degrees C showed three distinct peaks at 35.9, 39.7, and 49.1 degrees C and 34.5, 39.7, and 46.7 degrees C, respectively. The position of the largest peak for myosin remained unchanged, and the shift of the peak position of the highest temperature was about 3 degrees C, as obtained for the 10 degrees C-acclimated carp.(ABSTRACT TRUNCATED AT 250 WORDS)
Differential scanning calorimetry and CD spectrometry were employed to study the thermal unfolding of light meromyosin (LMM) prepared from carp acclimated to different temperatures. The transition temperatures given by the major peaks at pH 8.0 in 0.6 M KCl for LMM from carp acclimated to 10 degrees C were 32.5 and 39.5 degrees C with the calorimetric enthalpies (DeltaHcal) of 269 and 52 kcal/mol, respectively. LMM from carp acclimated to 20 degrees C exhibited three peaks of transition temperatures at 34.5, 40.2, and 46.9 with DeltaHcal of 152, 20, and 10 kcal/mol, respectively. On the other hand, LMM from carp acclimated to 30 degrees C showed two different patterns. The first experiment gave two transition temperatures at 39.2 and 47.3 degrees C with DeltaHcal of 231 and 39 kcal/mol, respectively. The second series of experiments resulted in showing three peaks of 34.4, 39.5, and 47.5 degrees C with DeltaHcal of 117, 123, and 28 kcal/mol, respectively. N-terminal amino acid sequence analysis revealed that LMM at the second series of experiments with the 30 degrees C-acclimated carp contained component(s) predominant in the 20 degrees C-acclimated carp. Thermal unfolding responsible for these transition temperatures was well explained by melting of alpha-helices which could be determined by far-ultraviolet CD spectroscopy. These results clearly demonstrate that the 30 degrees C-acclimated carp contained the most thermostable LMM.
The thermodynamic properties of myosin and its C-terminal fragment, light meromyosin (LMM), from walleye pollack, a typical cold-water fish efficiently utilized on an industrial scale, were analyzed by using differential scanning calorimetry (DSC) and circular dichroism (CD) spectrometry. Recombinant walleye pollack LMM expressed in Escherichia coli was also subjected to DSC and CD measurements for reference. The two proteins prepared from frozen surimi showed three endothermic peaks, the transition temperatures (T(m)) of which were quite similar, although overall DSC patterns differed considerably from one another. Their alpha-helical contents determined by CD were low compared to values reported before for other species. On the other hand, recombinant LMM gave four endothermic peaks at 27.4, 30.8, 36.5, and 43.4 degrees C in DSC and showed an alpha-helical content of approximately 80%. The peak at 27.4 degrees C could not be observed in walleye pollack LMM prepared from frozen surimi and thus was possibly attributed to its C terminus, because this extreme C-terminal region is supposedly truncated during preparation of LMM by tryptic digestion.
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