天花粉蛋白的化学 Ⅵ.用激光拉曼光谱研究天花粉蛋白的二级结构

由天花粉蛋白的水和重水溶液的激光拉曼光谱,测得酰胺Ⅲ谱带1240cm~(-1)和酰胺I谱带1632,1660cm~(-1)对CH_2弯曲模式1448cm~(-1)的强度比值。按Lippert等建立的方程组作定量计算,求得天花粉蛋白的二级结构含量为α-螺旋43.5％,β-折叠31.3％和无序25.2％,它们与4 分辨率天花粉蛋白单晶X射线衍射法的结果相一致。同时,研究了上述溶液的冻干粉状固体的二级结构,经过冻干,使其中约10％的β-折叠转变成无序构象,而α-螺旋含量无明显变化。在水溶液中,由测得的I_(850)/I_(830)比值计算,天花粉蛋白中的酪氨酸残基约有80％呈“暴露式”。     

天花粉蛋白的化学——Ⅴ.粉末状结晶天花粉蛋白的拉曼光谱

本文报道粉末状结晶天花粉蛋白的拉曼光谱。酰胺Ⅰ和酰胺Ⅲ的振动谱带在1680和1250 cm~(-1)。骨架C_α—C—N的振动谱带在965 cm~(-1)。苯丙氨酸残基的特征谱带在1010和1620 cm~(-1)。酪氨酸残基的特征谱带在845和862 cm~(-1)。色氨酸残基的特征谱带在765 cm~(-1)。酰胺Ⅰ和Ⅲ的谱带中,B折叠和无序结构的特征较为明显。表征无序结构的965 cm~(-1)谱带很弱。     

天花粉蛋白二级结构的圆二色性研究

本文报告了室温条件下在185—330nm波长范围内天花粉蛋白的CD谱测定的结果。利用205—240nm区间的CD谱,借助参考CD谱用最小二乘拟合的方法,计算得天花粉蛋白中α螺旋平均含量为24％,β折叠含量为30％,无序构象含量为46％。     

用概率统计法估算天花粉蛋白的二级结构

本文应用Levitt提出的蛋白质中氨基酸对二级结构的特定偏爱的统计方法估算天花粉蛋白的二级结构。计算结果表明,在天花粉蛋白中α-螺旋为32％,β-折叠为28％,反转为20％,其他类型为20％。这一计算结果与X射线衍射研究、圆二色性谱研究的结果相近。     

酰胺Ⅰ带和酰胺Ⅲ带测定花生磷脂酶D的α-螺旋和β-折叠含量

用傅立叶红外光谱的酰胺Ⅰ带和酰胺Ⅲ带测定花生磷脂酶D的α-螺旋和β-折叠含量.结果显示,两个谱带测定的β-折叠含量误差在3%以内,α-螺旋含量的误差在5%左右.     

天花粉蛋白分子的肽链走向

本文证明从4分辨率的电子密度图上确定了一条合理的肽走向。天花粉蛋白属于α β型结构。整个分子包含八段α-螺旋,约占残基总数37％;13条β链组成4个β折叠层,占残基总数32％。α-螺旋相对地处于分子的中心,而4个β折叠层分布在外围。这是天花粉蛋白结构的显著特点,这种结构排布方式尚未见文献报道。用坐标叠合方法获得了不对称单位内两分子叠合时的旋转矩阵和平移向量,叠合后的均方根误差是1.31。     

三七总皂甙对牛血清白蛋白溶液构象的影响

应用衰减全反射傅立叶变换红外光谱结合荧光光谱和紫外光谱研究了中药三七 的有效成分三七总皂甙与牛血清白蛋白（BSA）的相互作用，采用对蛋白质红外光 谱酰氨Ⅰ带和酰氨Ⅲ带进行曲线拟合的方法，定量分析了不同浓度三七总皂甙对 BSA二级结构的影响，发现随着三七总皂甙浓度的增加，蛋白分子结构逐渐发生了 由螺旋向折叠的转化。a-螺旋结构减少了3%，β-折叠结构增加了约5%，其它二级 结构没有明显的变化，红外差谱和荧光光谱的结果为药物与蛋白质的作用引起牛血 清白蛋白溶液构象的变化提供了佐证，紫外光谱反映了单体皂甙与蛋白质的结合常 数的差异。     

土壤埋藏对常见彩绘文物蛋白胶料形貌和结构的影响

利用扫描电子显微镜(SEM)、X-射线衍射法(XRD)和红外光谱法(FTIR)对猪皮胶、全蛋、蛋清、蛋黄、牛奶5种常见彩绘文物蛋白胶料老化前后的形貌、结晶度、红外光谱吸收特征和蛋白质二级结构含量进行了对比研究。结果表明,5种胶料老化后,表面规则的结构和纹理遭到破坏,整体变得松散;结晶度降低,尤以牛奶最显著;老化后,5种蛋白胶酰胺Ⅰ、Ⅱ、Ⅲ带红外吸收的阶梯状特征仍然明显,但螺旋结构已经解离,且均呈现α-螺旋含量降低、β-折叠和无规则卷曲含量增高的趋势,说明分子结构从有序趋于无序状态。     

红外光谱法分析酵母蛋白质的二级结构

采用红外光谱法分析了酵母蛋白质的二级结构。测定了不同温度下酵母酰胺Ⅲ带的一维红外光谱、二阶导数红外光谱及去卷积红外光谱。结果表明:随着测量温度的升高,酵母中的蛋白质α-螺旋结构的红外吸收强度降低;而β-转角结构、无规卷曲结构和β-折叠结构红外吸收强度均有所增加。还研究了酵母酰胺Ⅲ带的二维红外光谱,以确定酵母中蛋白质红外吸收强度的变化次序,进一步证明了酵母蛋白质的β-折叠结构的热不稳定性。     

八棱丝瓜蛋白1的二级结构及其生物活性

测定了八棱丝瓜蛋白1的N端顺序为Asp-Val-Ser-Phe-Ser-,用CD谱测定了八棱丝瓜蛋白 1的α螺旋,β-折叠和无规卷曲含量分别为37.1%,33.4%,29.5%。实验表明,八棱丝瓜蛋白 1具有RNA N-糖苷酶活性,体外抑制肿瘤细胞生长活性表明其对肿瘤细胞株B16,MGC,Bel的半数抑制浓度分别为1.78×10-7mol/L,2.11×10-7mol/L和4.21×10-7mol/L。并在N端顺序,二级结构和生物活性方面对八棱丝瓜蛋白 1和天花粉蛋白进行了比较。     

C-Terminal sequencing of trichosanthin

Preliminary identification of C-terminus of trichosanthin by chemical and enzymic methods, such as hydrazinolysis, thiohydantoin reaction and carboxypeptidase hydrolysis, showed that there may be the possible presence of more than one terminus, i.e., Met and Ala but complicated by side reactions. A computer-assisted carboxypeptidase method was first introduced by the authors to determine the C-terminal sequence of trichosanthin, and showed that trichosanthin is heterogeneous at its C-terminus and has two C-terminal sequences determined as -Arg-Asn-Asn-Met-OH and -Arg-Asn-Asn-Met-Ala-OH respectively. These results have been later unambiguously confirmed by the results from other experiments through the identification of the free alanine always present in the CNBr degradation products of trichosanthin, and the actual separation of two fragments from the finger prints as well as from the HPLC fractions of the trypsin digest of this protein. All shows that their amino acid sequences, determined by manual DABITC/PITC technique, agree well with those of the two C-terminal sequences determined by the computer-carboxy peptidase method.     

天花粉蛋白C-端顺序的测定

采用肼解法、乙内酰硫脲法和羧肽酶法检测了天花粉蛋白的C-端氨基酸有二,分别为Met和Ala借计算机辅助的羧肽酶法, 测定了天花粉蛋白C-端顺序, 其结果显示C-端是非均一的, 有两个未端, 分别为-ArgAsnAsnMetOH和-ArgAsnAsnMetAlaOH.这些结论已经从天花粉蛋白的溴化氰降解物中获得游离的Ala, 和从该蛋白的胰蛋白酶酶解物中分离得与该两顺序相符的两个碎片而完全证实.     

Chemistry of trichosanthin: VII. The chemical cleavage of the tryptophanyl peptide bond in trichosanthin

The single tryptophan residue of trichosanthin was cleaved by the use of BNPS-skatole reaction with STIG2 peptide fregment which was obtained from the tryptic digestion of N-succinyl trichosanthin. After separation, the N-terminal sequence of tryptophanyl C-side peptide was determined by use of manual DABITC-PITC double coupling sequencing method as follows: -Leu-Ala-Leu-Ser-Lys-Gln-Ile-Gln-Ile-Ala-.     

天花粉蛋白一级结构的修正及不同产地天花粉蛋白的研究

胰蛋白酶酶解天花粉蛋白, 用高效液相色谱分离酶解肽段, 用顺序仪测定其有关肽段的顺序。用羧肽酶A, B, Y测定了天花粉蛋白C-端和天花粉蛋白溴化氰降解肽CB1的C-端顺序, 修正了我们1985年测定的天花粉蛋白一级结构, 证明天花粉蛋白由246(7)氨基酸残基所组成, 除C-端微观不均一外, 与Collins结果一致。同时比较了芜湖产天花粉蛋白一级结构与平湖产的天花粉蛋白一级结构, 没有发现两者的一级结构有差别。     

The aggregation of trichosanthin in aqueous solution

As a kind of cytotoxin extracted from the root tuber of Trichosanthes kirilowii Maxim (Cucurbitaceae), trichosanthin can selectively bind to and kill the placental trophoblastic cells, which leads to a number of biomedical applications including the inhibition of trophoblastic tumors. However, the stability of trichosanthin in living organism is still one of the problems hindering the effectiveness of its applications. In this study, laser light scattering has been used successfully to investigate the stability of trichosanthin in both deionized water and KSCN aqueous solution in terms of the hydrodynamic size distribution of the trichosanthin aggregates as a function of both time and the salt concentration. It is found that the size distribution is always a bimodal one. One peak corresponds to a single trichosanthin chain; the other corresponds to the trichosanthin aggregates, which have an average hydrodynamic radius of ∼ 49 nm and are composed of ∼ 127 trichosanthin molecules when C_KSCN is higher than 0.5 mol/L. This implies that there exists an equilibrium between the single trichosanthin chain and its aggregates [i.e., nT ⇄ (T)_n]. Our results also suggest that the aggregates are made of the loosely packed trichosanthin molecules and behave as flexible polymer chains in θ solvent. © 1996 John Wiley & Sons, Inc.     

羟基蒽醌衍生物中羟基质子的核磁共振研究

通过对23个羟基蒽醌衍生物的质子核磁共振研究, 观察了不同取代类型羟基的化学位移范围;讨论了酚羟基乙酰化对芳环质子的化学位移影响, 为羟基取代位置的确定及芳环质子谱线归属提供了某种判据.此外, 还用HMO 法探讨了各类取代羟基的氢键强度及羰基迫位仅有一个羟基的内氢键强度与给予原子电荷密度的关系.本文报道一系列新型冠醚N,N'-双取代-1,7-二氧杂-12-冠-4(1~6), N,N' -双取代-1, 7-二氮杂-15冠-5(7~10) 及N,N'-双取代-1,10-二氮杂-18-冠-6(11~14)的质谱. 借助联动扫描和去焦技术对它们的开裂机理作了详细探讨.     

Chemical synthesis of a structural gene coding for trichosanthin

A structural gene (750 bp), which codes for a type I ribosome inactivating protein, trichosanthin, has been designed according to the codon usage of highly expressed gene in E. coli and chemically synthesized. In the synthesized gene, twenty-seven unique restriction sites were evenly dispersed with an average distance between two adjacent sites less than 50 bp to facilitate a systematic investigation on structure-functional relationship of this protein by site-directed muta-genesis. To synthesize it, the whole gene was divided into three large fragments (EP, PN and NH) which were assembled from several chemical synthetic oligonucleotides by enzymatic method. The assembly of both the fragment EP from six oligonucleotides (A-F) and the fragment PN from four oligomers (G-J) was catalyzed by T4 DNA ligase in using the single stranded DNA method [Chen, H.-B. et al, Nucl. Acids Res., 18, 871(1990)]. And fragment NH was formed from three duplexes K, L and M by the classical double stranded DNA method. Finally,     

X射线荧光光谱理论强度计算中激发因子的选择

激发因子是X射线荧光强度理论计算中的重要参数。对目前常用的四种激发因子算法进行了较为详细的比较,并设计了一种评价激发因子算法的实验方法,即用人工合成试样测定了八对能量接近的X射线特征谱线的强度比,然后根据不同的激发因子算法用理论方法计算出这些谱线对的强度比值,以两者的接近程度来判断这四种激发因子算法的适用性。结果表明,使用NRLXRF程序修改版中的激发因子算法计算出来的理论强度比与实测值之间的相对偏差最小。