Kaca W, Roth R I, Vandegriff K D, Chen G C, Kuypers F A, Winslow R M, Levin J
Department of Laboratory Medicine, University of California School of Medicine, San Francisco, USA.
Biochemistry. 1995 Sep 5;34(35):11176-85. doi: 10.1021/bi00035a024.
Previous investigations have demonstrated that hemoglobin (Hb) is a binding protein for bacterial endotoxin (lipopolysaccharide, LPS) and that the structure and biological activity of LPS are altered in the presence of Hb. In the present study, the influence of LPS on the structure of native human HbA0 and covalently cross-linked Hb (alpha alpha Hb) was studied by analyzing the absorption and circular dichroic spectra of Hb in the wavelength region of 200-650 nm. Incubation of oxyHb with each of several LPSs resulted in a decrease in the intensity of the major Soret band at 414 nm with a shift in the maximum peak to 410 nm, decreases in the intensities of the major visible region peaks at 541 and 577 nm, and the appearance of increased absorbance in the visible region in the range of 630 nm. The resultant spectra are characteristic of methemoglobin formation. These spectral changes were time-dependent and LPS-concentration-dependent. Production of methemoglobin was prominent with chemically modified, partially deacetylated rough LPS, and was observed to a lesser extent both with native, complete rough and with native smooth LPSs. The influence of LPS on the absorption spectrum of methemoglobin also was directly tested. The conversion of methemoglobin to hemichrome in the presence of LPS was demonstrated and was shown to be reversible. Analysis of circular dichroic spectra of Hb demonstrated LPS-induced spectral changes in the visible and Soret regions consistent with the production of a substantial quantity of metHb, but did not demonstrate any alteration in the far-UV region (210-240 nm). Moreover, Hb oxygen affinity was only slightly altered after incubation with any of several LPSs. In conclusion, analyses of absorption and circular dichroic spectra reveal the potential of LPS to produce a facilitated oxidation of both alpha alpha-cross-linked human Hb and native human HbA0, without substantial changes in the secondary structure of the globin.
先前的研究表明，血红蛋白（Hb）是细菌内毒素（脂多糖，LPS）的结合蛋白，并且在Hb存在的情况下，LPS的结构和生物活性会发生改变。在本研究中，通过分析Hb在200 - 650 nm波长区域的吸收光谱和圆二色光谱，研究了LPS对天然人HbA0和共价交联Hb（ααHb）结构的影响。将氧合血红蛋白与几种LPS分别孵育后，414 nm处主要Soret带的强度降低，最大峰位移至410 nm，541和577 nm处主要可见区峰的强度降低，并且在630 nm范围内的可见区出现吸光度增加。所得光谱是高铁血红蛋白形成的特征。这些光谱变化是时间依赖性和LPS浓度依赖性的。化学修饰的部分脱乙酰化粗糙LPS产生高铁血红蛋白的现象很明显，而天然的完全粗糙LPS和天然光滑LPS产生高铁血红蛋白的程度较小。还直接测试了LPS对高铁血红蛋白吸收光谱的影响。结果表明，在LPS存在下高铁血红蛋白向半色原的转化是可逆的。对Hb圆二色光谱的分析表明，LPS在可见区和Soret区引起的光谱变化与大量高铁血红蛋白的产生一致，但在远紫外区（210 - 240 nm）未显示任何变化。此外，与几种LPS中的任何一种孵育后，Hb的氧亲和力仅略有改变。总之，吸收光谱和圆二色光谱分析表明，LPS有可能促进αα交联的人Hb和天然人HbA0的氧化，而球蛋白的二级结构没有实质性变化。
