tailieunhanh - Bioluminescence Recent Advances in Oceanic Measurements and Laboratory Applications Part 10

Tham khảo tài liệu 'bioluminescence recent advances in oceanic measurements and laboratory applications part 10', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | 126 Bioluminescence - Recent Advances in Oceanic Measurements and Laboratory Applications investigated pH range of Table 2 . Neither x12 nor UG showed any significant widening of the spectra at lower pH. Overall bioluminescence spectra of x12 were even less pH-dependant than those of UG. Both thermostable mutants would be expected to retain their colour under physiological conditions which may be pertinent to multispectral imaging. The tolerance of x12 Fluc to low pH may give it further advantage in terms of signal strength. A Luciferase activity 20 pl at pM was assayed by the manual mixing with 180 pl of TEM mM ATP 222 pM LH2 300 pM CoA over a range of pH values between and . Bioluminescence was integrated over 5 s using the luminometer at a PMT voltage of 550 mV normalised to each luciferase enzyme total activity data A and non-normalised data B . Measurements for UG were carried out using a PMT voltage of 700 mV to obtain good signal-to-noise ratio readings data presented for the non-normalised curve have been corrected for the different PMT voltage used. The lag-time between initiation of the reaction and recording of light emission was 5s. Measurements at each pH were carried out in triplicate. Error bars represent one . within triplicate measurements. Fig. 3. Effect of pH on the activity of WT UG x5 and x12 Fluc. Kinetic parameters including Michaelis-Menten constants Km and catalytic constant Kcat were measured for the WT x12 and UG luciferases with LH2 and ALH2. The Kms of x12 Fluc pM and UG pM for LH2 were lower than that of WT Fluc 18 pM Table 3 indicating an increase in affinity of the thermostable enzymes for LH2 relative to the WT. Even stabilising mutations far from the active site can affect tertiary structure and lead to increased substrate affinities and altered enzyme kinetics Squirrell et al. 1999 . Catalytic efficiency characterised by Kcat Km ratio was significantly compromised in both thermostable .