Effect Of Hifu On Lysozyme Structure And Function

BACKGROUNDHigh intensity focused ultrasound (HIFU) is a new technology of Micro noninvasive treatment which has exhibited a great advantage in clinical applications. The action mechanism of HIFU includes thermal effect, mechanical effect and cavitation effect, especially the research on cavitation effect has been one of the focus of all fields. Until recently, there are few reports about HIFU irradiation effects on protein structure or function. Therefore, we use lysozyme as a model protein to discuss this problem, which is solved by several means in this paper such as mass spectrometry, circular two color spectrum, fluorescence spectrum, nuclear magnetic resonance hydrogen spectrum, polyacrylamide gel electrophoresis and passive cavitation detection. And find out the mechanism of HIFU irradiation in protein structure and function.OBJECTIVE:1. To investigate the effect of HIFU on the lysozyme molecular structure.2. To investigate the effect of HIFU on the lysozyme molecular function.3. To study the cavitation phenomenon of HIFU in the radiation process.4. To study the focal region temperature variations of HIFU in the radiation process.METHODS:1.The lysozyme solution was exposed at HIFU with pulse frequency 0.94 MHz, sound intensity 3.25×104 W/cm2, pulse repetition period 4 Hz and du ty cycle 10% for 10 s,2min,3min,4min,5min. The lysozyme solution was exposed at HIFU with pulse frequency 0.94 MHz, pulse repetition period 4 Hz and duty cycle 10% for 5 min, using Irradiation power 100W,200W, 300W,400W,500W.2.The effect of HIFU on lysozyme molecular structure was studied using C D spectra, Fluorescence spectrum,1H nuclear magnetic resonance (1H-NM R) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) method.3. In the process of HIFU irradiation,using the PCD system collect the focal area of acoustic emission signal (acoustic emissions, mainly for the cavitation signal), after the fast fourier transformation of the signal (Fast Fourier Transformation, FFT),we can get the harmonic changes with the irradiation time. Duing to the signal is band-pass filter (3-7MHz), to calculate the root mean square (Root Mean Square, RMS), and get the curve of RMS change with irradiation time. Then get the accumulated cavitation dose irradiation (ICD).4. Using temperature acquisition system to record the focal region temperature in the HIFU irradiation, and draw standard curve..RESULTS:1.The Result showed that after HIFU treatment, there was no obvious chan ge in CD spectrumindicating that HIFU might cause little effect on lysozy me lower structure. Whereas a weaken fluorescence intensity appeared whi ch related to lysozyme conformation change. Data of SDS-PAGE showed o bvious degradation of lysozyme branches.1H-NMR analysis demonstrated that tryptophan (Trp) residue of lysozyme displayed chemical shift after tre atment.2. As for the activity of lysozyme, it was lower in all the HIFU treatment groups.3. During HIFU irradiation, cavitation phenomenon, and with the increase of irradiation power, the cumulative dose of cavitation increased.4. During HIFU irradiation, the focal region temperature variations little, which will not cause the degeneration.CONCLUSIONS:1.The study confirmed that the advanced structure of lysozyme (third and fourth structure) could be changed by HIFU, while primary structure (protein primary sequence and secondary structure) did not change obviously.2. After HIFU irradiation, the activity of lysozyme reduced, meanwhile, a longer irradiation time and power, a weaker increasement in the activity growth.3. There appears cavitation phenomenon during irradiation, free radicals including free hydroxyl hydrogen atom which are produced by cavitation, will effect the electric distribution of the protein surface that lower the protein activity.