Lepock J R, Frey H E, Inniss W E
Guelph-Waterloo Program for Graduate Work in Physics, University of Waterloo, Canada.
Biochim Biophys Acta. 1990 Oct 15;1055(1):19-26. doi: 10.1016/0167-4889(90)90086-s.
Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax. The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively. For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megaterium and B. psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.
采用差示扫描量热法（DSC）分析嗜热脂肪芽孢杆菌（ATCC 12016和WAT）、嗜温巨大芽孢杆菌及嗜冷嗜冷芽孢杆菌这三种菌株中的热转变。将观察到的代表脂质熔化以及DNA和蛋白质解折叠的转变，与每个物种的最高生长温度（Tmax）进行比较，以此来确定导致热诱导生长抑制的关键、热不稳定靶点。在嗜热脂肪芽孢杆菌和巨大芽孢杆菌中检测到一个低温脂质转变，该转变随Tmax略有变化，但其高温端总是比Tmax低22 - 33摄氏度。DNA主要熔化的转变温度（Tm）在88至92摄氏度之间，比Tmax高23 - 32摄氏度。代表不可逆转变的图谱主要部分可分解为至少三个不同的峰，并且主要被确定为蛋白质变性。变性起始温度（Tl），即可检测到变性的最低温度，在一定程度上取决于生长温度（Tg）。嗜热脂肪芽孢杆菌ATCC和WAT的Tmax分别为69摄氏度和56摄氏度。对于在比Tmax低4至20摄氏度之间生长的细胞，Tl比Tmax低2 - 4摄氏度，这表明在生长完全抑制之前可以耐受一些变性，并且表明生长抑制是由于Tm比Tl高几度的关键蛋白质变性，或者是由于变性蛋白质积累到临界水平。对于巨大芽孢杆菌和嗜冷芽孢杆菌也有类似模式，只是Tmax分别为48摄氏度和32.5摄氏度（Tl分别为45 - 46摄氏度和30摄氏度）。因此，对于同一属中的这三个物种，蛋白质变性起始与最高生长温度之间存在极好的相关性。本研究还证明了DSC在基于细胞成分的热稳定性解析完整细胞中的转变以及获得大分子稳定性整体视图方面的适用性。
