Abstract：The effect of different temperatures on the activity and conformational changes of proteinase K was studied. Methods Proteinase K was treated with different temperatures, then denatured natural substrate casein was used to assay enzyme activity, steady-state and time-resolved fluorescence spectroscopy was used to study tertiary structure, and circular dichroism was used to study secondary structure. Results show with the temperature rising from 25 to 65 ℃, the enzyme activity and half-life of proteinase K dropped, maximum emission wavelength red shifted from 335 to 354 nm with fluorescence intensity decreasing. Synchronous fluorescence intensity of tryptophan residues decreased and that of tyrosine residues increased. Fluorescence lifetime of tryptophan residues reduced from 4.427 1 to 4.032 4 ns and the fraction of α-helix dropped. It was concluded that it is simple and accurate to use steady-state/time-resolved fluorescence spectroscopy and circular dichroism to investigate thermal stability of proteinase K. Thermal denaturation of proteinase K followed a three-state process. Fluorescence intensity of proteinase K was affected by fluorescence resonance energy transfer from tyrosine to tryptophan residues. The α-helix was the main structure to maintain conformational stability of enzyme active site of proteinase K.
张奇兵，那馨竹，尹宗宁* . 光谱法表征蛋白酶K的热变性过程 [J]. 光谱学与光谱分析, 2013, 33(07): 1749-1753.
ZHANG Qi-bing, NA Xin-zhu, YIN Zong-ning* . Characterization of Thermal Denaturation Process of Proteinase K by Spectrometry . SPECTROSCOPY AND SPECTRAL ANALYSIS, 2013, 33(07): 1749-1753.
[1] Devaraj K B, Kumar P R, Prakash V. Process Biochemistry, 2011, 46: 458. [2] Tripathi T, Rseler A, Rahlfs S, et al. Biochimie, 2010, 92: 284. [3] Halder U C, Chakraborty J, Das N, et al. Journal of Photochemistry and Photobiology B: Biology, 2012, 109: 50. [4] Jany K D, Lederer G, Mayer B. Febs Letters, 1986, 199(2): 139. [5] Liu S Q, Tao Y, Meng Z H, et al. Journal of Molecular Modeling, 2011, 17: 289. [6] Tao Y, Rao Z H, Liu S Q. Journal of Biomolecular Structure & Dynamics, 2010, 28(10): 143. [7] Tomar R, Dubey V K, Jagannadham M V. Biochimie, 2009, 91: 951. [8] Pechkova E, Tripathi S, Ravelli R B G, et al. Journal of Structural Biology, 2009, 168: 409. [9] MA Chun-qi, LI Ke-an, ZHAO Feng-lin, et al(马春琪，李克安，赵凤林，等). Acta Chimica Sinica(化学学报), 1999, 57: 389. [10] Fraga A S, Esteves A C, Micaelo N, et al. International Journal of Biological Macromolecules, 2012, 50: 323. [11] Cui L, Du G C, Zhang D X, et al. Bioresource Technology, 2008, 99: 3794. [12] Sultan N A M, Swamy M J. Journal of Photochemistry and Photobiology B: Biology, 2005, 80: 93. [13] Kuznetsova I M, Stepanenko O V, Stepanenko O V, et al. Biochemistry, 2002, 41: 13127.
