Dong A, Nagai M, Yoneyama Y, Caughey W S
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523.
J Biol Chem. 1994 Oct 14;269(41):25365-8.
The reduced abnormal subunits of two M-type hemoglobins, Boston (His alpha 58-->Tyr) and Saskatoon (His beta 63-->Tyr), have been determined in the presence of normal human hemoglobin A by measurement of C-O stretch bands in infrared spectra of carbon monoxide complexes. Use of an infrared microscope coupled to a Fourier transform infrared spectrometer of high sensitivity permitted measurements to be made on as small a hemoglobin mixture as is contained in a single erythrocyte. The abnormal subunits of both Hbs M exhibit bands near 1970 cm-1 compared with bands near 1951 cm-1 for the normal subunits. The increase in 1970 cm-1 band intensity upon erythrocyte reduction with dithionite provided a measure of the extent of abnormal subunit oxidation; in cell suspensions about 60% of the abnormal subunits of Hb M Boston and 80% for Hb M Saskatoon remained reduced. The amount of Hb present as abnormal Hb averaged about 25% for Hb M Boston cells and about 50% for Hb M Saskatoon cells. However, the ratio of Hb M to Hb A in individual cells varied markedly, with the ratio expected to decrease as the cell ages. These results demonstrate the unique utility of infrared microspectroscopy for the study of differences in abnormal Hb status among individual erythrocytes.
通过测量一氧化碳复合物红外光谱中的C - O伸缩带，在正常人血红蛋白A存在的情况下，对两种M型血红蛋白（波士顿型（Hisα58→Tyr）和萨斯卡通型（Hisβ63→Tyr））的还原异常亚基进行了测定。使用与高灵敏度傅里叶变换红外光谱仪相连的红外显微镜，能够对单个红细胞中所含的极少量血红蛋白混合物进行测量。与正常亚基在1951 cm-1附近的谱带相比，两种M型血红蛋白的异常亚基在1970 cm-1附近均呈现谱带。用连二亚硫酸盐使红细胞还原后，1970 cm-1谱带强度的增加提供了异常亚基氧化程度的一种度量；在细胞悬液中，波士顿型M血红蛋白约60%的异常亚基和萨斯卡通型M血红蛋白80%的异常亚基仍保持还原状态。以异常血红蛋白形式存在的血红蛋白量，波士顿型M血红蛋白细胞平均约为25%，萨斯卡通型M血红蛋白细胞约为50%。然而，单个细胞中M型血红蛋白与A 型血红蛋白的比例变化显著，预计该比例会随着细胞老化而降低。这些结果证明了红外显微光谱法在研究单个红细胞中异常血红蛋白状态差异方面的独特用途。
