大白鼠糖致白内障的激光喇曼光谱研究

作者用激光喇曼光谱法分析半乳糖导致大白鼠晶状体混浊过程中构象的变化。通过SPEX 1403型激光喇曼光谱仪得到了正常及不同混浊度晶状体的喇曼光谱。结果表明晶状体可溶性蛋白质二级结构的光谱未见异常,其残基酪氨酸及色氨酸微环境起了变化。随着晶状体混浊度的增加,SH谱峰强度变小而S-S键谱峰增强,同时观察到荧光背景逐渐加强。经分析认为晶状体混浊是与蛋白质分子的聚集有关。     

胆石中胆红素钙的FTIR定量分析

 应用付立叶变换红外光谱(FT-IR)测定胆石中胆红素钙的含量,使用KBr压片法,吸收度是由积分法表示。胆红素钙在1622.3cm~(-1),1253.1cm~(-1)等处有特征吸收峰,在FT-IR减谱分析的基础上,选定1253.1cm~(-1)为定量吸收峰,它符合Beer-Lambert’s定律(r=0.998)而且共存物干扰小。标准工作曲线是使用胆红素为标准。胆石样品中胆红素钙含量用此法测定,其结果与化学法结果相似。应用FT-IR对混合物定量分析简单、迅速、准确。     

双股寡聚d(G-C)6 DNA Z-构象的形成及其构象转换的动态研究

研究揭示,生物信息的形成,传递与DNA构象的多样性,特别是其中的左手螺旋Z-构象DNA（Z-DNA）相关.在机体DNA链中,普遍存在的特异序列结构d（C-G）n和d（G-C）n片段易形成Z-构象.但对d（G-C）n序列结构的寡聚体Oligo-d（G-C）n,（n小于8）能转换形成Z-DNA片段少见报道.为促进对Z-DNA尤其是其中的短片段Z-DNA与生物功能的相关性研究,我们对合成并纯化后的寡聚体Oligo-d（G-C）n,n分别为4,6,8,10, 及Oligo-d（C-G）6和多聚体poly-d（G-C）500-900进行Z-构象的形成和其构象转换的比较研究.研究结果发现：①d（GpCpGpCpGpCpGpCpGpCpGpC）是d（G-C）n序列结构中能转换形成Z-构象的最短片段（n＝6）.其转换成Z-构象能力有链长依赖性（poly d（G-C）500-900易于Oligo-d（G-C）6）;②Oligo-d（G-C）6的Z-构象形成能力因溶液中的介质性质不同而异.Co（NH3）3＋〉Mg2＋〉Na＋;C1O-4〉Cl-,因此要求盐溶液的浓度差异很大.③PH7.2,室温条件下,在MgCl2, NaClO4, NaCl溶液浓度分别由0 mol/L增至6.0 mol/L,Oligo-d（G-C）6的B、Z构象转换都出现：B-构象相对稳定期,B-、Z-构象转换跃迁期和Z-构象相对稳定期.每个阶段要求跨越的盐浓度变迁范围也因所用介质而异.当溶液中Oligo-d（G-C）6 B-构象、Z-构象各占50%（θ1/2）时,其盐浓度分别为1.72 mol/L（MgCl2）,2.88 mol/L（NaClO4）,3.85 mol/L（NaCl）.④Oligo-d（G-C）6的B-,Z-构象转换程度受盐浓度影响：当Oligo-d（G-C）6处于最适条件和不同盐溶液其浓度为θ（12）浓度时,温度由8 ℃→22 ℃,在MgCl2,NaClO4溶液中的Oligo-d（G-C）6形成Z-构象能力增加,当由22 ℃→60 ℃,MgCl2溶液中的Z-构象Oligo-d（G-C）6加速增加,而在NaClO4溶液中则是急速向B-型Oligo-d（G-C）6方向转换;温度变化对处于NaCl溶液中的Oligo-d（G-C）6B-、Z-构象相对平衡影响较小.⑤甲基化胞嘧啶即Oligo-d（G-mC）6或d（mC-G）6均增大Z-构象形成能力.⑥在4 mol/L MgCl2溶液中的Oligo-d（G-C）6或Oligo-d（C-G）6或poly d（G-C）500-900的UVab谱、UVcd谱均显示出非B-型或Z-型DNA的新谱型.并且有链长依赖性和因溶液浓度改变出现构象可逆性转变.提示在Oligo-d（G-C）6的构象转换过程中可能存在新构象＂X＂型,即BZX构象转换模式.     

质粒DNA超螺旋构象的激光喇曼光谱研究

将pBR322重组质粒DNA纯化,经琼脂糖凝胶电泳鉴定其主要具共价闭合环状空间构象。对此制备物进行激光喇曼散射光谱分析,发现在表征其二级结构为B型的特征模之外,还有另外二个表征磷酸脱氧核糖主链骨架振动状态的特征模854和1083cm~(-1)。本文对此进行探讨,认为这二个特征模与闭合环状DNA分子的超螺旋状态有关,可作为质粒DNA三级结构的特征模。碱基堆积状态分析表明,超螺旋的存在使分子中脱氧胸苷的堆积反应活性增强,并使AT碱基间Hoogsteen型氢键有相当数量的破坏,导致反映脱氧胸苷参与氢键组成的二个基团振动状态的特征模1378cm~(-1)产生相对于线性DNA分子的明显减色及脱氧胸苷羰基双键振动模向高波数偏移。     

水稻病毒激光喇曼光谱的初步探讨

本文初步探讨水稻病毒激光喇曼光谱,分析了谱线与病毒中核酸蛋白质结构的关系。确信应用激光喇曼光谱研究水稻病毒是具有信息量大、高灵敏及高分辨本领的。     

细胞色素bc_1复合物的喇曼光谱研究

对提纯的细胞色素ｂｃ１复合物的氧化态和底物琥珀酸还原态两个样品进行了共振喇曼和富立叶喇曼光谱测定和比较。琥珀酸还原态与氧化态的共振谱比较明显有变化,而富立叶红外谱没有什么差别。说明呼吸链的电子传递体在氧化态与还原态交替变化进行电子传递时,蛋白总体构象不发生大的改变,而活性中心血红素辅基局部构象变化很大。     

博莱霉素-Ce(Ⅲ)对DNA的切断机理初探

根据外切核酸酶Ⅲ酶解博莱霉素-Ce(Ⅲ)［BLMA5-Ce(Ⅲ)］作用过的双链直线型DNA时, 酶解速率明显增大, 酶解产物除5′-dAMP、5′-dGMP、5′-dCMP和5′-dTMP 4种单核苷酸外, 还有其他成分存在的实验事实, 推测出BLMA5-Ce(Ⅲ)在DNA双链的特定部位沿5′→3′的方向切断磷酸二酯键, 使DNA的双链上形成多个暴露的3′-OH末端.     

钙调蛋白的结构与功能(下)

四、钙调蛋白的构象 CaM分子本身无酶的活性,在无Ca~(2+)情况下,也无生物活性。自从1973年Teo等人鉴定它是一种钙结合蛋白以来,人们用各种物理、化学手段发现Ca_(2+)的结合引起蛋白强烈的构象变化,Ca~(2+)·CaM复合物才能与靶蛋白结合形成一个活性全酶,因此CaM构象问题是其调控机制的中心问题。 1.研究构象的方法 (1)圆二色性与紫外差谱的研究 Klee测定CaM的远紫外CD谱,在EGTA存在下,椭圆度[Q]_(221nm)=-11500度·cm~2/分·克分子,相当35％螺旋,50％无规卷曲,20％β-折迭。而在0.3mM Ca~(2+)存在下,[Q]_(221nm)增加约20％,相当于α螺旋增加5—8％,无规卷曲相     

不同pH条件下细菌视紫红质的共振拉曼光谱研究

本实验测定了不同pH条件下嗜盐菌紫膜中细菌视紫红质(bR)的共振拉曼光谱.13-顺式视黄醛生色团的特征峰1187cm~(-1)和全反式、13-顺式共有的特征峰1200cm~(-1)带强度之比I_(1187)/I_(1200)在pH1.0-8.9之间约为0.76,而pH高于8.9为0.97.pH3.0-9.0时C=NH~ 振动峰为1640-1642cm~(-1),pH9.4以上为1642-1644cm~(-1),pH9.2附近变化最大,pH3.0以下低于1640cm~(-1).酸性和弱碱性范围时,19-CH_3和20-CH_3的面内变形振动与面外变形振动相互重叠,碱性范围分为双峰.并讨论了对结构及其稳定性的影响.     

马槟榔甜味蛋白的圆二色谱和激光拉曼光谱

测定了自马槟榔(Capparis masaikai Levl.)种子分离的二种甜味蛋白MaⅠ和MaⅡ的远紫外区域圆二色谱,并按Yang和Chen的方法用最小二乘法计算了它们的构象单元含量,结果表明α螺旋含量最多:对MaⅠf_H=0.43、f_β=0.24、f_R=0.33;对MaⅡ,f_H=0.37、f_β=0.33、f_R=0.30;其相关系数均为0.9876;计算所得理论曲线与实验曲钱其本吻合。二种甜蛋白的激光拉曼光谱测定结果也表明其主要构象单元为α螺旋,此外,均无SH谱带:MaⅠ的Tyr残基暴露于分子表面;与MaⅡ相比,MaⅠ有明显的545cm~(-1)和1101cm~(-1)谱带,这可能从构象上说明MaⅠ与MaⅡ对热和变性剂处理表现不同的原因。     

Raman spectroscopy of interferon-induced 2',5'-linked oligoadenylates

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.     

Energetics of Z-DNA formation in poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T).

The conformational change for the alternating purine-pyrimidine polydeoxyribonucleotides i.e. poly d(A-T), poly d(G-C), and poly d(A-C) poly d(G-T) from a right-handed conformation at room temperature to the left-handed Z-DNA like double helix at elevated temperatures has been studied by UV spectroscopy, Raman spectroscopy, and by adiabatic differential scanning microcalorimetry (DSC) in the presence of Na+ and Mg2+ or Ni2+ respectively as counterions. The differential UV spectra reveal through a hyperchromic shift at around 280nm and a hypochromic shift at 260nm that a conformational change to the left-handed conformation occurs. The Raman spectra clearly show characteristic changes, a drastic decrease of the band at 680cm-1 and the appearance of a new band at 628cm-1, due to the change of the purine bases to the syn conformation upon inversion of the helix-handedness. The course of the transition as function of temperature can be followed quantitatively by plotting the change in the excess heat capacity vs. temperature. The transition enthalpy delta H for the B- to Z-DNA transition per mole base pairs (mbp) amounts to 2.0 +/- 0.2kcal for poly d(G-C), to 4.0 +/- 0.4kcal for poly d(A-T), and to 3.1 +/- 0.3kcal for poly d(A-C) poly d(G-T). The enthalpy change due to the Z-DNA to coil transitions (per mole base pairs) amounts to 11kcal for poly d(G-C), 10.5kcal for poly d(A-T) and 11.3kcal for poly d(A-C) poly d(G-T).     

Effect of the G.T mismatch on backbone and sugar conformations of Z-DNA and B-DNA: analysis by Raman spectroscopy of crystal and solution structures of d(CGCGTG) and d(CGCGCG)

The Z-DNA crystal structures of d(CGCGTG) and d(CGCGCG) are compared by laser Raman spectroscopy. Raman bands originating from vibrations of the phosphodiester groups and sensitive to the DNA backbone conformation are similar for the two structures, indicating no significant perturbation to the Z-DNA backbone as a result of the incorporation of G.T mismatches. Both Z structures also exhibit Raman markers at 625 and 670 cm-1, assigned respectively to C3'-endo/syn-dG (internal) and C2'-endo/syn-dG conformers (3' terminus). Additional Raman intensity near 620 and 670 cm-1 in the spectrum of the d(CGCGTG) crystal is assigned to C4'-exo/syn-dG conformers at the mismatch sites (penultimate from the 5' terminus). A Raman band at 1680 cm-1, detected only in the d(CGCGTG) crystal, is assigned to the hydrogen-bonded dT residues and is proposed as a definitive marker of the Z-DNA wobble G.T pair. For aqueous solutions, the Raman spectra of d(CGCGTG) and d(CGCGCG) are those of B-DNA, but with significant differences between them. For example, the usual B-form marker band at 832 cm-1 in the spectrum of d(CGCGTG) is about 40% less intense than the corresponding band in the spectrum of d(CGCGCG), and the former structure exhibits a companion band at 864 cm-1 not observed for d(CGCGCG). The simplest interpretation of these results is that the conventional B-form OPO geometry occurs for only 6 of the 10 OPO groups of d(CGCGTG). The remaining four OPO groups, believed to be those at or near the mismatch site, are in an "unusual B" conformation which generates the 864 cm-1 band.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman spectroscopic studies of the DNA cro binding site conformation, free and bound to cro protein.

Raman spectra of the DNA binding site for cro repressor protein were obtained in the presence and absence of bound cro protein. The 17 base pair fragment is a consensus sequence of the six cro binding sites in phage lambda, except that the second base to the right of the center of pseudosymmetry is altered. Analysis of the spectrum of the free DNA indicates that the molecule exists in a B-like conformation with deviations from the usual B form occurring mainly in the bands assigned to A-T vibrations. The spectrum of the bound DNA was obtained by subtracting the spectrum of free cro from the spectrum of the complex which was estimated to be 90% bound. The DNA undergoes significant structural changes upon binding to the protein; most notable of these changes is a destacking of the G-C bases reflected by increases in the 1240, 1262, and 1320 cm-1 bands. A decrease in the 1361 cm-1 band that occurs has also been assigned to a destacking in guanine bases. The appearance of a 705 cm-1 band and the decrease and downshift of the 670 cm-1 band are consistent with the appearance of A-like character in the A-T region of the binding site when the protein binds; however, the spectra indicate that the entire binding site remains in a distorted B-like conformation. We use the 705 cm-1 band to estimate A-like character because the 800-850 cm-1 region is obscured by interference from strong protein bands. Other shifts in both intensity and position cannot be assigned to characteristic changes in conformation and therefore must be attributed to the protein influencing the structure in a novel way.     

A Raman scattering study of the helix-destabilizing gene-5 protein with adenine-containing nucleotides.

Raman spectra of gp5 and complexes of gp5 with poly(rA) and poly(dA) have been determined and analysed. From a fit of the amide I-band with model spectra it follows that the secondary structure of gp5 contains 52% beta-sheet, 28% undefined conformation and 19% alpha-helix. The band at 1032 cm-1 due to phenylalanine has an anomalous intensity both in the spectra of the complexes and the free protein. This possibly indicates a stacked structure present in the protein. Binding of gp5 to poly(rA) and poly(dA) influences the intensity of bands near 1338 and 1480 cm-1 which are considered to be marker-bands for the phosphate-sugar-base conformer. A change in conformation of the nucleotides is also reflected by vibrations originating in the phosphate- and sugar-residues of the backbone. In the spectrum of complexed poly(rA) the intensity of the conformation sensitive band at 813 cm-1, which is due to the phosphodiester group, is zero. It seems that gp5 forces poly(rA) and poly(dA) to a similar conformation. A marker band for stacking interaction in poly(rA) indicates that stacking interactions in the complex have increased.     

Intensity changes of base and backbone Raman modes in the B to Z transition of poly(dG-dm5C)

Raman spectroscopy was employed to investigate the temperature-induced B to Z transition of poly(dG-dm5C). The transition midpoint was about 37 degrees C for a solvent containing 20 mM Mg2+. A 10-fold change in Mg2+ concentration altered the transition midpoint by at least 60 degrees C. Raman spectra of the B and Z forms of poly(dG-dm5C) exhibited characteristics similar to those observed with poly(dG-dC). The 682 cm-1 guanine mode and 835 cm-1 backbone mode were present in the B conformation. In the Z form the intensities of these two bands decrease substantially and new peaks were observed at 621 cm-1, 805 and 819 cm-1. Several bands unique to poly(dG-dm5C) were also observed. Transition profiles of band intensity vs. temperature were determined for fourteen Raman bands. The curves of all of the base vibrations and one backbone mode had the same slope and midpoint. This indicates that conformational changes in the guanine and methycytosine bases occur concurrently.     

Sequence dependence of conformations of self-complementary duplex tetradeoxynucleotides containing cytosine and guanine

The four self-complementary tetradeoxynucleotides which contain only cytosine and guanine are 5'-d-(CpGpCpG)-3', 5'-d(CpCpGpG)-3', 5'-d(GpCpGpC)-3', and 5'-d(GpGpCpC)-3'. The Raman spectra of aqueous solutions (about 0.05 M in monomer) of these tetranucleotides at pH 7 and 2 degrees C show clearly that these self-complementary tetranucleotides form double-stranded duplex structures of the canonical B type when the NaCl concentration is 0.5 M NaCl. If the temperature is raised to 50 degrees C, the Raman spectra show that in each case the double-helical B form melts in a non-cooperative way to a disordered single-chain form. On the other hand, if the salt concentration is raised to saturation, the Raman spectrum of only one of these four tetranucleotide solutions at 2 degrees C is changed in any substantial way. The Raman spectrum of the tetranucleotide 5'-d(CpGpCpG)-3' at 2.2 degrees C and at 4 M or higher salt concentration strongly resembles that of double-helical Z-form poly(dC-dG) taken under similar conditions. We conclude that the tetramer 5'-d(CpGpCpG)-3' is the only self-complementary double-helical tetranucleotide containing only cytosine and guanine in which the B-Z transition can be induced by increasing the salt concentration. This tetramer has several types of stacking interactions which differ markedly from stacking interactions in the other tetramers and may account for the enhanced stabilization of its Z conformation.     

Chemical synthesis of 5'-pyrophosphate and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. CD study of the effect of 5'-phosphate groups on the conformation of 3'-5' GpG.

A simple, two-step method is described for the synthesis of the 5'-pyro- and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. The readily accessible 2'(3')-5' ApA, ApG, GpA and GpG were converted in one step to the corresponding 5'-phosphoramidate derivatives which were then transformed to the 5'-pyro- and triphosphates. CD spectra of 3'-5' pn GpG (n = 0,1,2 or 3) derivatives, measured at pH 1, indicated stabilization of the (syn) G+p (anti)G conformation by the 5'-phosphate groups.     

Raman spectroscopic study of left-handed Z-RNA

The solvent conditions that induce the formation of a left-handed Z form of poly[r(G-C)] have been extended to include 6.5 M NaBr at 35 degrees C and 3.8 M MgCl2 at room temperature. The analysis of the A----Z transition in RNA by circular dichroism (CD), 1H and 31P NMR, and Raman spectroscopy shows that two distinct forms of left-handed RNA exist. The ZR-RNA structure forms in high concentrations of NaBr and NaClO4 and exhibits a unique CD signature. ZD-RNA is found in concentrated MgCl2 and has a CD signature similar to the Z form of poly[d(G-C)]. The loss of Raman intensity of the 813-cm-1 A-form marker band in both the A----ZR-RNA and A----ZD-RNA transitions parallels the loss of intensity at 835 cm-1 in the B----Z transition of DNA. A guanine vibration that is sensitive to the glycosyl torsion angle shifts from 671 cm-1 in A-RNA to 641 cm-1 in both ZD- and ZR-RNA, similar to the B----Z transition in DNA in which this band shifts from 682 to 625 cm-1. Significant differences in the glycosyl angle and sugar pucker between Z-DNA and Z-RNA are suggested by the 16-cm-1 difference in the position of this band. The Raman evidence for structural difference between ZD- and ZR-RNA comes from two groups of bands: First, Raman intensities between 1180 and 1600 cm-1 of ZD-RNA differ from those for ZR-RNA, corroborating the CD evidence for differences in base-stacking geometry. Second, the phosphodiester stretching bands near 815 cm-1 provide evidence of differences in backbone geometry between ZD- and ZR-RNA.