Phage T4 lysozyme. Physical properties and reversible unfolding.
Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07-1 g/l the c-m equals 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c-m equals 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10-4 M dithiothreitol and 10-6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T-m, increment H-unf and increment S-unf with increasing pH.
D001665:Binding Sites; D002942:Circular Dichroism; D003090:Coliphages / Q000201:enzymology*; D004267:DNA Viruses / Q000201:enzymology; D004229:Dithiothreitol; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D009113:Muramidase; D009905:Optical Rotatory Dispersion; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012965:Sodium Chloride; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D006146:Guanidines; D012965:Sodium Chloride; D009113:Muramidase; D004229:Dithiothreitol