藻胆体发射终端能量传递过程的研究

利用超快速时间分辨光谱研究了蓝绿藻藻胆体的２个变藻蓝发射终端（ＡＰＢ,ＡＰＩ）单体和三聚体内的能量传递过程,探测到ＡＰＢ和ＡＰＩ单体的两组亚基α＾ＡＰ／β＾ＡＰ和α＾ＡＰＢ／β＾ＡＰ间的能量传递时间常数分别为３０ｐｓ和１９４ｐｓ。ＡＰＢ和ＡＰＩ三聚体所共有的９－３２ｐｓ的短寿命组分来源于同一单体内能量由α＾ＡＰ向βＡＰ的传递过程。     

阴离子交换和羟基磷灰石色谱从螺旋藻中提纯藻胆蛋白的研究

本文采用离子交换层析和羟基磷灰石吸附层析技术从螺旋藻中提取纯化得到藻蓝蛋白和别藻蓝蛋白。通过比较 ＡＮＸ Ｓｅｐｈａｒｏｓｅ ４ Ｆａｓｔ Ｆｌｏｗ（ｈｉｇｈ ｓｕｂ／ｌｏｗ ｓｕｂ）、 ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ和 Ｑ Ｓａｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ等阴离子交换树脂的动态吸附容量以及目标产品的纯度,选用 ＤＥＡＥＳｅｐｈａｒｏｓｅ Ｆａｓｔ Ｆｌｏｗ作为层析介质．对离子交换的产物进行了电泳分析,藻蓝蛋白和别藻蓝蛋白的等电点接近,电迁移速率相似。采用羟基磷灰石吸附技术对藻胆蛋白混合物进一步分离纯化,分别得到了藻蓝蛋白和别藻蓝蛋白的纯品,经等电聚焦实验验证显示为均一组成。     

A transient absorption study of allophycocyanin

Transient dynamics of allophycocyanin trimers and monomers are observed by using the pump-probe, transient absorption technique. The origin of spectral components of the transient absorption spectra is discussed in terms of both kinetics and spectroscopy. We find that the energy gap between the ground and excited states of the unexcited subunit of allophycocyanin monomer decreases via an interaction with another excited subunit. For allophycocyanin trimer, we find that the fast dynamics results from the fast internal conversion and the first excited state is the only one electronic state which can trap the final population.     

光谱学方法对钝顶螺旋藻异藻蓝蛋白活性构象研究(英文)

采用羟基磷灰石层析和离子交换层系的方法从钝顶螺旋藻(Spirulina platensis)中分离纯化出异藻蓝蛋白。对异藻蓝蛋白光谱性质受溶液pH变化的影响做了研究。异藻蓝蛋白吸收光谱峰值在650 nm,在620 nm处有1个肩峰,荧光激发光谱的最大发射波长在660 nm。以紫外-可见光吸收光谱表征异藻蓝蛋白对光能的吸收能力,以荧光光谱表征其光能传递能力,以圆二色谱表征其二级结构变化。光谱分析结果表明异藻蓝蛋白在pH为4 ～10的范围之内,其光能吸收和传递的能力保持在一个相对较稳定的范围内,同时异藻蓝蛋白维持三聚体的形式,而其二级结构则会发生一定程度扰动。在pH值低于4或高于10的范围内,异藻蓝蛋白三聚体发生解聚,二级结构发生剧烈变化,其光能吸收和传递能力迅速被破坏。     

Improved biocompatibility of protein encapsulation in sol–gel materials

By using the fluorescent dye 6-propionyl-2-(N,N-dimethylamino) naphthalene (PRODAN) to monitor methanol generated during tetramethyl orthosilicate polymerization we have optimised the encapsulation of protein in silica sol–gel monoliths with respect to completion of hydrolysis and distillation in order to remove methanol such that protein can be added without denaturation. A minimum of 24 h at +4 °C was found to be required before hydrolysis is complete and 3–5 min of vacuum distillation at 50 °C and 300 mbar needed to remove methanol before the gel is formed. The biocompatibility of a tetramethyl orthosilicate sol–gel monolith was demonstrated by preserving the trimer protein allophycocyanin (APC) in its native form for up to 500 h. This obviates the previously essential requirement of covalently binding the trimer together in order to prevent dissociation into monomers and has enabled observation of native APC trimer in a sol–gel pore for the first time down to the single molecule level using combined fluorescence spectroscopy and confocal microscopy. The higher stability afforded by the protocol we describe could impact on the application of sol–gel materials to single-molecule studies of wider bearing such as protein folding and aggregation.     

Labeling of cytosine residues with biliproteins for use as fluorescent DNA probes

The fluorescent properties of biliproteins (B-phycoerythrin, BPE; C-phycocyanin, CPC and allophycocyanin, APC) have been utilized as labels of nucleic acids to detect hybridization by means of steady-state fluorescence anisotropy in a homogeneous aqueous solution of model system in which poly(C) and poly(I) are, respectively, the probe and target sequences. An easy method to obtain biliprotein-DNA conjugates by a two-stage procedure is described. The first stage was a modification of the cytosine amino group of poly(C) at the N⁴ position which then reacted with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to obtain poly(C)-bound 2-pyridyl disulfide. In the second stage biliproteins were directly reacted with SPDP to obtain biliprotein-bound 2-pyridyl disulfide, dithiotreitol was added for reduction to biliprotein-bound thiol and was then mixed with the poly(C)-bound 2-pyridyl disulfide to obtain the biliprotein-poly(C) conjugate. The three biliproteins studied bind to nucleic acids without noticeable change of their spectral properties (absorption, fluorescence efficiency and fluorescence lifetime). Consequently, our labeling methodology can be applied to obtain any type of biliprotein-labeled nucleic acid probe. The increase of the steady-state fluorescence anisotropy from biliprotein-poly(C) upon hybridization with poly(I) can be used to readily detect the hybridization with the target poly(I) in a sample without having to separate free and bound labeled probes. The small decreases in lifetime displayed by the biliprotein-poly(C) conjugates upon hybridization are not sufficient to explain the steady-state anisotropy increase. Apparently, the rotational motion of the overall macrostructure is principally responsible for the increase in anisotropy. The greater fluorescence efficiency and lifetime from BPE with respect to those from CPC or APC allows us recommend that protein as the most suitable label.