氘標記拉曼顯微光譜在活體酵母菌之研究

Abstract

拉曼顯微光譜儀是一個適合研究活體細胞新陳代謝的強而有力工具。而且當我們結合同位素標記的分子時，它便可以有效地、更深入地去追蹤細胞新陳代謝時的分子動態，不久前我們實驗室曾發表：透過13C標記的葡萄醣餵食酵母菌，使同位素進入活體細胞，這一次我們改用全部氘〈2H〉取代葡萄醣延伸這項實驗，由於氘的質量數是氫的兩倍，可以預期將表現更大的同位素取代效應，在以氘取代葡萄醣為主要碳來源下進行一系列的培養時間解析實驗，脂質、細胞質〈蛋白質豐富的區域〉與細胞壁〈多醣〉之拉曼光譜，經過氘的同位素取代後，C-H伸縮震動波峰〈3000—2800 cm-1〉向低頻區位移到2150 cm-1附近，有別於先前的研究多半專注在指紋區的變化，其光譜範圍為1800—800 cm-1，C-D伸縮震動波峰的消長是相當地顯而易見，其出現的區段原先並沒有任何拉曼波峰〈silent region〉，所以透過C-D伸縮震動峰來追蹤新陳代謝不會被其他震動波峰干擾。此外除了C-D伸縮震動波峰增強外，我們還發現在蛋白質充沛的區域下測得的光譜中，phenylalanine殘基的ring breathing震動模式1003 cm-1，紅位移到976 cm-1, 961 cm-1，為了解釋這樣的同位素取代現象，我們透過理論計算去預測當殘基六碳環上的氫被取代成氘時的所有可能性，並且與實驗值去比較對照，在這次的研究裡也將呈現多變數分析的拉曼影像。

Raman microspectroscopy provides powerful means to investigate cellular metabolism in living cells. When coupled with stable isotope labeling, it becomes even more powerful owing to the additional ability to trace intracellular metabolic dynamics. Recently we have demonstrated this novel approach using 13C-labeled glucose. In this study, we extend our work to deuterium (2H) labeling, which exhibits the largest isotope substitution effect because of a twofold difference in atomic mass. We measured a series of space-resolved Raman spectra from lipid droplets and the cytoplasmic (protein-rich) and cell wall (polysaccharide-rich) regions in living yeast cells at different incubation times in medium containing perdeuterated glucose as the primary carbon source. Upon 2H-isotope substitution, the C-H stretch band shifts from 3000–2800 to 2150 cm−1. Unlike other Raman bands in the congested fingerprint region (800–1800 cm−1), the C-D stretch band is easily discernible because it appears in the silent region, thereby facilitating the tracing of the time-dependent changes without ambiguity. In addition to the rise of the C-D stretch band, the ring breathing mode of the phenylalanine residues in proteins at 1003 cm−1 is found to downshift to two different wavenumbers, i.e., 961 and 975 cm−1. In order to explain this isotope substitution effect, we compare the experimental results with theoretical calculation results of the phenylalanine molecule whose ring hydrogen(s) are substituted with deuterium. We also present and discuss single-cell Raman imaging results.