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采用交换吸附脱色及柱层析新技术,成功地从烟叶中分离和纯化了一种烟草过氧化物酶同工酶(TOPI),并对其进行了一系列光谱特性研究。基质辅助激光解吸电离飞行时间质谱(WALDI-TOF-MS)测得TOPI分子量为21888.5,等电点pI为3.5;氨基酸组成分析表明TOPI为一含血红素的酸性蛋白酶。光谱学分析揭示,在402nm处有一典型的Soret带,在498和636nm处有特征吸收峰(α,β带),酸度和变性剂对TOPI在紫外可见区的特征吸收峰均产生一定的影响,TOPI的荧光光谱测定结果说明TOPⅠ分子中含有Trp残基和Tyr残基以及其自身的独特的光谱学特性。
A novel tobacco peroxidase isoenzyme (TOPI) was successfully isolated and purified from tobacco leaves using a series of new spectroscopic techniques, including exchange-exchange decolorization and column chromatography. The TOPI molecular weight was 21888.5 and the isoelectric point pI was 3.5 by matrix-assisted laser desorption ionization time of flight mass spectrometry (WALDI-TOF-MS). Amino acid composition analysis showed TOPI was a heme-containing acidic protease. Spectroscopic analysis revealed a typical Soret band at 402 nm with characteristic absorption bands (α, β bands) at 498 and 636 nm. The acidity and denaturant agents have some influence on the peak absorption of TOPI in the UV-visible region. The results of the fluorescence spectrometry of TOPI indicate that the TOPI molecule contains the unique spectral properties of Trp residues and Tyr residues as well as its own.