Probing pheomelanin synthesis using thioflavin T fluorescence
Probing pheomelanin synthesis using thioflavin T fluorescence
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DOI：
10.1117/12.2506381
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发表时间：
2019-03
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影响因子：
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通讯作者：
A. Davy;D. Birch
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作者：
A. Davy;D. Birch
荧光转子探针硫黄素T（ThT）通过研究由L - 半胱氨酸、L - 多巴和酪氨酸酶合成的褐黑素的形成与分解，用于探究褐黑素中片状结构的存在。棕色/黑色的真黑素和红色/黄色的褐黑素是黑色素最常见的形式，前者被更广泛地研究。数十年研究的结论是，两种黑色素都具有不明确的聚合物结构，这反映在它们复杂的光物理性质上。这两种形式的结构完整性和共存对它们的功能有重要影响，实际上它们的光谱学为作为黑色素瘤生物标志物提供了可能性。外在荧光探针很少用于研究黑色素，但来自ThT荧光的证据与其他技术一致，发现真黑素的组成成分二羟基吲哚形成类似于石墨的堆叠片状结构。对于褐黑素的组成成分苯并噻嗪和苯并噻唑是否存在类似的片状结构尚未确定。在研究这种可能性时，我们发现褐黑素合成过程中ThT荧光呈S形增加，这与在真黑素中观察到的情况相似，意味着存在片状结构。褐黑素中ThT荧光的红移以及出现约5.5纳秒的荧光衰减成分（比ThT本身的长得多），是根据ThT与褐黑素天然结构耦合的位点来解释的。当黑色素被激发时，这些位点可以作为荧光陷阱，能量被汇聚到此处，从而提高了复合荧光产率。然而，我们发现这种衰减成分在褐黑素中的含量（6%）明显低于真黑素（11%），这表明ThT不太容易嵌入褐黑素。这可能反映出褐黑素的片状结构较少或其片层结合更紧密，这与其低荧光量子产率是一致的。提高pH值分解褐黑素提供了进一步的光谱学证据，表明ThT被困在较小寡聚形式的片状聚集体中。
The fluorescent rotor probe thioflavin T (ThT) is used to investigate the presence of sheet structures in pheomelanin by studying the formation and disassembly of pheomelanin synthesized from L-cysteine, L-DOPA and the enzyme tyrosinase. Brown/black eumelanin and red/yellow pheomelanin are the most common forms of melanin with the former more extensively studied. The conclusion of decades of research is that both melanins possess ill-defined polymeric structures, which are reflected in their complex photophysics. The structural integrity and co-existence of these two forms has significant bearing on their functionality and indeed their spectroscopy offers possibilities as a melanoma biomarker. Extrinsic fluorescence probes have been little used to study melanin, but the evidence from ThT fluorescence is consistent with other techniques in finding that eumelanin’s constituent dihydroxyindoles form a stacked sheet structure akin to graphite. Whether or not such an equivalent sheet structure exists for pheomelanin’s constituent benzothiazines and benzothiazoles is unresolved. On investigating this possibility we find a sigmoidal increase in ThT fluorescence during pheomelanin synthesis is similar to that observed for eumelanin, implying a sheet structure. A red spectral shift in ThT fluorescence in pheomelanin and the emergence of a fluorescence decay component of ~ 5.5 ns, much longer than that of ThT, are interpreted in terms of sites wherein ThT is coupled to pheomelanin’s native structure. Such sites can act as fluorescent traps to which energy is funneled when melanin is excited and the composite fluorescence yield thus increased. However, we find the abundance of this decay component is significantly less for pheomelanin (6%) than eumelanin (11%), suggesting that ThT does not intercalate so readily in pheomelanin. This may reflect less sheet structure or tighter binding of pheomelanin’s sheets, consistent with its low fluorescence quantum yield. Disassembling pheomelanin upon raising pH provides further spectroscopic evidence that ThT is trapped in sheet-like aggregates of smaller oligomeric forms.