一种从鱿鱼提取色素的方法<em> Doryteuthis pealeii</em
Summary
一种颜料从纳米颗粒鱿鱼Doryteuthis pealeii色素细胞提取协议提出。
Abstract
Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.
Introduction
头足类如鱿鱼，墨鱼，章鱼必须动态改变其为伪装和信令外观的能力。1-6这种能力部分地由被称为色素细胞色素器官的选择性面积扩张支撑。4,7-9色素细胞是包含一个cytoelastic球囊内局限于纳米色素颗粒是由肌纤维径向固定网络软执行器。1,3-当他们被启动，色素细胞500％中提出表面积整个器官分配颗粒扩大3,7 ，10,11当该动作一致在若干色素细胞的，动物的整体着色被改变。而，已知的颜料颗粒有助于这种颜色的变化，它们的组合物仍然不明。我们描述了一种方法分离和纯化可被适于将来组成研究色素细胞色素。
<p claSS =“jove_content”>颜料颗粒的隔离涉及多步骤提取，同质化和纯化过程。3,12含有色素细胞的组织是通过从头足类小心摘除收获。然后将消化和均化过程用于解离周围组织并分离色素细胞的细胞。纳米结构的颗粒然后使用重复超声处理和离心剩余的色素细胞分离和纯化。经纯化，颜料是从一个过程中的颗粒是从由蝴蝶翅膀可见颜色的提取使用酸性甲醇溶液适于萃取。13扫描电子显微镜（SEM）和分光用于确认色素细胞色素使用成功地提取这一过程。该方法描述了一种用于探索到c的分子贡献色素细胞颗粒的隔离oloration在头足类动物。从整体动物12小分子的提取往往是一个漫长而乏味的过程。这里的目的是为了通知有效和容易的协议的未来研究人员为购自于头足类动物的纳米颗粒颜料。
Protocol
Representative Results
Discussion
我们已经证明的方法来提取鱿鱼色素细胞颗粒颜料。通过专门针对颗粒，我们的目标是，以确定其在调节适应性着色作用。这种方法使用批量组织样本14或冷冻干燥的皮肤从15到设计的头足类动物的特征色素以前的报告不同。
虽然该协议是在提取色素细胞色素有效，它被限制为小反应的鳞片。解剖1鱿鱼产量〜11毫克纯化的颗粒。约该质量的58％含有12可…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.
Materials
| Collagenase | Alfa Aesar | 9001-12-1 | No hazard |
| Dithiothreitol | Sigma-Aldrich | 3482-12-3 | Irritant, acute toxicity |
| HEPES | Sigma-Aldrich | 7365-45-9 | Mild irritant |
| Hydrochloric acid | EMD Chemicals | 7647-01-0 | Corrosive |
| k-Aspartate | Sigma-Aldrich | 1115-63-5 | Reacts violently with oxidants |
| Magnesium Chloride | Sigma-Aldrich | 7786-30-3 | Mild eye irritant |
| Methanol | Pharmco-AAPER | 67-56-1 | Highly flammable |
| Mini tablet prtoease inhibitor | Sigma-Aldrich | 469315-90-01 | Corrosive to metal and skin |
| Papain | Sigma-Aldrich | 9001-73-4 | Irritant |
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