Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study.
Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study.
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人补体蛋白 C9 的热解折叠和聚集：差示扫描量热研究。
DOI：
10.1021/bi00240a035
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Esser,AF
中科院分区：
文献类型：
--
作者：
Lohner,K;Esser,AF
材料与方法：根据Esser和Sodetz（1988）的描述，从过时的血浆或Cohn分数III膏体中分离出人类C9。考马斯蓝染色蛋白的SDS-PAGE分析或放射碘化蛋白的放射自显影表明，分离蛋白的纯度优于99%，未检出蛋白水解片段。无钙C9 （apo-C9）按照前面描述的方法制备（Thielens等人，1988），或者简单地通过在相同的缓冲液中添加1mm EDTA来生产。用凝血酶原水解法制备了Nicked C9 (C9n) （Dankert et al., 1985）。所有样品在4℃下透析过夜，进入指定的缓冲液，最后的透析缓冲液用于参考测量，以提供基线热图。在室温下调节所有缓冲液的pH值。在量热测量之前，蛋白质溶液在贝克曼离心机中常规离心，以去除在透析或储存期间可能形成的任何聚集体。采用消光系数为9.6 mg/(mL* cm)的280 nm分光光度法测定C9的最终浓度，通常为1mg /mL （Esser & Sodetz, 1988）。差示扫描量热法。除非另有说明，否则使用Microcal MC-2D仪器（Microcal Inc., Amherst， MA）在60℃/h的加热速率下进行量热测量。细胞用N2加压至1.5 atm以防止加热时冒泡，仪器在4℃下热平衡约1小时后开始扫描。热量计使用12位模拟/数字转换板（Data Translation DT-2801）与IBM-AT计算机连接，用于自动数据采集。采用厂家提供的DA-1软件包进行数据采集和分析。简单地说，在对存储的缓冲基线进行子提取后，通过将每个数据点除以扫描速率和量热计电池中C9的摩尔数，将原始数据（每分钟毫卡路里）归一化为多余的热容量曲线（千卡路里每摩尔开尔文），然后进行数字滤波以去除短期噪声而不会失真峰值形状。这些热像图随后被用于进一步的模拟分析。该软件提供了四种不同的子程序来模拟实验数据：(1)假设所有转变都是独立的两态转变，AHvli= AHal·；(2)假设独立的非两态转变，HvH不等于Ha]·；(3)假设Freire和Biltonen（1978）讨论的顺序两态转变；(4)假设具有ACp的独立两态转换。
Materials and MethodsProteins. Human C9 was isolated either from outdated plasma or from Cohn fraction III paste as described by Esser and Sodetz (1988). SDS-PAGE analysis of Coomassie Blue stained protein or autoradiography of radioiodinated protein indicated that the isolated protein was better than 99% pure and no proteolytic fragments could be detected. Calcium-free C9 (apo-C9) was prepared as described previously (Thielens et al., 1988) or simply produced by addition of 1 mM EDTA to the same buffer. Nicked C9 (C9n) was prepared by pro-teolysis with-thrombin (Dankert et al., 1985). All samples were dialyzed overnight at 4 C into the indicated buffer, and the final dialysis buffer was used in reference measurements to provide base-line thermograms. The pH of all buffers was adjusted at room temperature. Before the calorimetric mea-surements, the protein solutions were routinely centrifuged in a Beckman Airfuge to remove any aggregates that may have formed during dialysis or storage. The final C9 concentration of C9, usually about 1 mg/mL, was determined spectropho-tometrically by using an extinction coefficient of 9.6 mg/(mL* cm) at 280 nm (Esser & Sodetz, 1988). Differential Scanning Calorimetry. A Microcal MC-2D instrument (MicroCal Inc., Amherst, MA) was used for calorimetric measurements at a heating rate of 60 C/h unless otherwise indicated. The cells were pressurized with N2 to 1.5 atm to preventbubbling on heating, and the instrument was thermally equilibrated at 4 C forabout 1 h before the scan was started. The calorimeter was interfaced to an IBM-AT computer using a 12-bit analog/digital conversion board (Data Translation DT-2801) for automatic data collection. The DA-1 software package provided by the manufacturer was used for data acquisition and analysis. Briefly, after sub-straction of the stored buffer base line, the raw data (milli-calories per minute) were normalized to excess heat capacity curves (kilocalories perdegrees kelvin per mole) by dividing each datapoint by the scan rate and by the number of moles of C9 in the calorimeter cell and then digitally filtered to remove short-term noise without distortion of the peak shape. These thermograms were then used for further simulation analyses. The software provides four different subroutines to simulate the experimental data:(1) assumes all transitions are independent two-state transitionswith AHvli= AHal·,(2) assumes independent non-two-state transitions, with HvH not being equal to Ha]·,(3) assumes sequential two-state transitions as discussed by Freire and Biltonen (1978);(4) assumes an independent two-state transition with a ACp.
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[1] 扫描量热法
DOI：
--
发表时间：
1978
期刊：
影响因子：
--
作者：
K. Krishnan;J. Brandts
通讯作者：
J. Brandts
聚(C9) 的分子量。
DOI：
10.1016/s0021-9258(17)43495-8
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
J. Tschopp;A. Engel;E. Podack
通讯作者：
E. Podack
补体裂解：目标羊红细胞膜上 C5b-9 复合物的超微结构和取向
影响因子：
3.7
作者：
Jørgen Tranum;S. Bhakdi;B. Bhakdi;Ole J. Bjerrum;V. Speth
通讯作者：
V. Speth
DOI：
10.1016/s0021-9258(19)57427-0
发表时间：
1988-01
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
R. Discipio;D. Chakravarti;H. J. MULLER-EBERHARD;G. Fey
通讯作者：
R. Discipio;D. Chakravarti;H. J. MULLER-EBERHARD;G. Fey
人类补体第九种成分的疏水结构域的光标记。
DOI：
--
发表时间：
1982
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Ishida,B;Wisnieski,BJ;Lavine,CH;Esser,AF
通讯作者：
Esser,AF