SPECTROPHOTOMETRY OF INTRACELLULAR RESPIRATORY PIGMENTS
SPECTROPHOTOMETRY OF INTRACELLULAR RESPIRATORY PIGMENTS
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DOI：
10.1126/science.120.3124.767
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发表时间：
1954-01-01
期刊：
影响因子：
56.9
通讯作者：
CHANCE, B
中科院分区：
文献类型：
--
作者：
CHANCE, B
具有化学特异性、能快速测量生物化学过程并能在活细胞上进行观察的物理学方法是罕见的，而光谱复制直到最近才发展到可以广泛研究各种物质的程度。对细胞内酶系统整合作用的观察是对深入的生物化学研究的必要补充，这些研究成功地将细胞内酶系统分解成可溶性组分，并在溶液中重建了许多重要的生物化学过程。但是，这些重建的成功和从它们发展而来的理论只能通过与体内系统的直接测量进行比较来评估。在这种情况下，物理方法仅限于生物化学物质，这些物质的吸收光谱响应于生物功能而改变足够的量，以允许令人满意的测量。在哺乳动物细胞呼吸酶的序列中，我们可以详细地研究六种组分的反应。表1给出了六种成分最满意测量的波长。其他不随代谢活动而改变吸收的细胞色素可通过适当的方法进行补偿，使其不干扰对活性色素的研究。方法. Mac-Munn（1）、Keilin（2）、瓦尔堡（3）和日本工作者（4）详细研究了可见光谱中细胞色素的可见光谱。这种方法对于快速鉴定各种微生物中的细胞色素类型仍然非常有用。但此法最适合于细胞色素a、B带的研究（650至500英里）;位于440到400 μ m区域中的5到10倍强的Y带不能通过可见光谱学有效地研究（5），也不能对还原的吡啶核苷酸（340 μ m）进行任何测量。对微生物细胞色素的光谱研究显示它们的Y带（6），但是在这些较短波长处的较大光散射效应叠加在光吸收上，从而给出相当失真的记录。密立根在1937年的工作（7）指出了我们目前的仪器灵敏，选择性和快速记录活细胞悬浮液和组织中微小的光密度变化的方法。他以非常简单的形式使用了两个基本特征：（1）一个微分色度计（遵循廷德尔原理，8），它只对两个紧密间隔的波长带的吸收变化做出反应，其中一个波长带位于待研究的色素吸收带的峰值;（2）一个生物系统，它可以存在于两个明确定义的状态中，并且可以快速和随意地从一个状态过渡到另一个状态。这样，两束光的相对强度在一种状态下被调节为零响应，生物系统被转移到另一种状态，细胞色素的吸收量被直接记录下来。
P YHYSICAL muethods that are chemically specific, that are fast enough to measure biochem-ical processes, and that permit observations to be made on living cells are rare, and spectros-copy has only recently been developed to the point where extensive studies of a wide range of material are possible. Such observations of the integrated ac-tion of intracellular enzyme systems are essential complements to the intensive biochemical researches that have succeeded in breaking down the cellular en-zyme systems into their soluble components and in re-constructing many important biochemical processes in solution. But the success of these reconstructions and the theories that evolve from them can be evaluated only by comparison with the direct measurements of the in vivo system. The physical method is limited in this case to biochemical substances that change their absorption spectra a sufficient amount in response to biological function to permit a satisfactory measure-ment. In the sequence of respiratory enzymes of mam-malian cells we can study in detail the reactions of six components. Table 1 gives the wavelengths at which the six components are most satisfactorily measured. Other cell pigments that do not change their absorp-tion in response to metabolic activity are compensated by suitable methods so that they do not interfere with the study of the active ones. Methods. Visual spectroscopy of cell pigments in the visible spectrum has been done in detail by Mac-Munn (1), Keilin (2), Warburg (3), and Japanese workers (4). This method is still extremely useful for the rapid identification of the types of cytochrome in various microorganisms. However, thismethod is best suited for the study of the a and, B bands of cyto-chromes (650 to 500 mi); the 5-to" 10-fold stronger y bands that lie in the region 440 to'400 mt cannot be studied effectively by visual spectroscopy (5), nor can any measurements be made of reduced pyridine nu-cleotide (340 m>).Spectrog-taphic studies of cytochromes of microorganisms revealed their y bands (6), but the larger light-scattering effects at these shorter wavelengths were superimposed upon the light absorption to give a considerably distorted record. Millikan's work (7) in 1937 pointed the way to our current instruments for the sensitive, selective, and rapid recording of small optical density changes in living cell suspensions and tissues. He used in very simple form two essential features:(i) a differential colorimeter (following Tyndall's principle, 8) that was responsive only to a change in absorption at two closely spaced wavelength bands, one at the peak of the absorption band of the pigment to be studied;(ii) a biological system that could exist in two clearly defined states, and in which the transition from one to the other could be made rapidly and at will. Thus the relative intensities of the two light beams are adjusted initially to give zero response in one state, the biological system is shifted to the other state, and the magnituade of the absorption of the cell pigment is recorded directly.