Denaturation of lysozyme and myoglobin in laser spray.
In laser spray, the tip of an electrospray capillary is irradiated with a continuous CO(2) laser beam. Here, we report results from a modified laser spray method that employs a relatively low laser irradiance level. With a laser power of approximately 2 W and a focal spot size ( approximately 0.3 mm), which covered the entire front surface of the electrospray capillary, the irradiance was approximately 3 x 10(3) W/cm(2). This resulted in a quiescent and smooth vaporization of aqueous solutions. This "evaporation-mode" laser spray method yielded the best results so far obtained in our laboratory with laser-irradiated electrospray, producing higher and more stable signals. The method was applied to the analysis of aqueous solutions of lysozyme and myoglobin. Mass spectra were obtained as a function of laser power from 0 W (electrospray) to approximately 2 W. The spray generated at the tip of the stainless steel capillary was observed with a CCD camera. With increase of laser power, the droplets in the spray became finer and the Taylor cone became progressively smaller. The strongest ion signals were recorded when the sample solution protruded only slightly from the tip of the capillary. A broadening of the lysozyme charge-state distribution, attributable to protein unfolding, was observed with a laser power of 2 W. No denaturation of myoglobin took place up to a laser power of 1.6 W. However, a sudden onset of denaturation was observed at 1.8 W as a broadening of the myoglobin charge distribution and the appearance of apo-myoglobin peaks. These findings demonstrate that laser spray is capable of dissociating the noncovalent complexes selectively without breaking covalent bonds.
Takamizawa
Atsushi
A
Clean Energy Research Center, University of Yamanashi, Takeda 4-3-11, Kofu 400-8511, Japan.
Fujimaki
Susumu
S
Sunner
Jan
J
Hiraoka
Kenzo
K
D000818:Animals; D002645:Chickens; D004527:Egg Proteins / Q000737:chemistry; D006736:Horses; D007834:Lasers; D008956:Models, Chemical; D009113:Muramidase / Q000737:chemistry*; D009211:Myoglobin / Q000737:chemistry*; D011489:Protein Denaturation; D021241:Spectrometry, Mass, Electrospray Ionization
D004527:Egg Proteins; D009211:Myoglobin; D009113:Muramidase