~1H SPIN SYSTEM IDENTIFICATIONS OF S-SULFONATED INSULIN B CHAIN WITH 2D-NMR

Resonance assignments of the ~1H spectrum of insulin are the basis on which to investi-gate its solution conformation by using NMR method. Owing to the complicated aggregationbehaviour of the molecule to give broadened n. m. r. lines, only limited resonance assignmentshave been reported. S-sulfonated A and B chains of insulin gave ~1H spectra with good reso-lutions. Based on the 500 MHz absolute 2D-COSY spectrum and 400 MHz phase sensitiveDQF-COSY, Relayed-COSY and NOESY spectra of B chain recorded in D_2O, all of thespin system identifications of the non-labile protons in the S-sulfonated B chain of insulinwere reported including the specific resonance assignments of eight residues: B_3Asn, B_9Ser,B_(16)Tyr, B_(22)Arg, B_(26)Tyr, B_(27)Thr, B_(28)Pro and B_(29)Lys. The pK values of B_(16) and B_(26) tyrosineare 10.65 and 10.60 respectively from pH titration.     

以双量子谱解析胰岛素类似物中氨基酸残基质子自旋体系

氨基酸残基质子自旋体系解析,是以核磁共振方法研究蛋白质等生物大分子结构、功能、运动的基础。二维双量子谱是解析残基质子自旋体系的重要手段。对B链C端去五肽胰岛素及S-磺酸型胰岛素A链,分别在水中及重水中测定其500 MHz相敏双量子谱和300 MHz相敏双量子谱。由双量子谱的直接峰、远程峰、等价峰,解析各类残基质子自旋体系,确认了从COSY谱所作的解析,提供了可供解析的更多信息。     

A CIRCULAR DICHROIC STUDY OF THE INTERACTION OF INSULIN A AND B CHAINS

It has been shown previously that the S-thiomethyl forms of the insulin A and B chains interact in solution leading to a partial transfer of Tyr side chains from hydrophilic to hydrophobic environments. In the present work, a circular dichroic study has indicated that the α-helix contents of the chains show a gradual increase upon mixing of the chains reaching completion in about 2h. The separated S-thiomethyl protected chains contain some ordered structure (A: α=15%, β=27%: B: α=22%, β=23%). Contrary to reports in the literature, the reduced chains also show some ordered structure and upon mixing of the reduced chains, the α-helix content also shows some increase. The ordered structure of the reduced chains decrease with increasing concentrations of dithiothreitel and in presence of a large excess of DTT both the reduced chains have very little, if any, α-helix structure. These latter results are in accord with those of Wu and Yang. In agreement with the results obtained previously with ultravio     

胰岛素类似物氨基酸残基质子自旋体系的二维谱模拟

以MARS程序对胰岛素类似物的残基质子自旋体系作计算机模拟,由模拟谱与B链C端去五肽胰岛素、S-磺酸型胰岛素A、B链的二维相关谱的比较,表明模拟谱与实测谱交叉峰图形及精细结构十分相似,验证了实测谱所作残基质子自旋体系的鉴别,同时也表明此程序可广泛应用于蛋白质等生物分子的二维谱模拟。     

胰岛素A和B链相互作用的圆二色性研究

本文报道以圆二色性方法在相同条件下测得S-硫甲基型A链的α螺旋、β折迭的含量为15％及27％;B链为22％,23％;A和B链混合后为24％,26％。表明A和B链相混后肽链构象有一定调整,α螺旋含量有所增加。以还原型A及B链进行实验,也得到类似结果。加入二巯基苏糖醇明显减少A及B链的有序成分。上述结果表明胰岛素A及B链有一定二级结构;在能重组成胰岛素的最适条件下,A及B链相混后,因次级键相互作用,两肽链构象有一定调整,有序程度有所提高。     

以二维核磁共振解析S-磺酸型胰岛素B链残基质子自旋体系

胰岛素~1H谱的谱峰识别,是以核磁共振方法研究其溶液构象的基础。由于胰岛素复杂的聚合行为,使谱峰变宽,难以作谱峰识别。S-磺酸型胰岛素A及B链有分辨良好的氢谱。本文报道以500MHz二维相关谱(绝对值型)、400MHz双量子滤波相关谱、接力相干转移相关谱、核Overhauser二维谱,对S-磺酸型B链在重水中~1H谱的残基质子自旋体系的解析;报道了B_3Asn,B_9 Ser,B_(16)Tyr,B_(22)Arg,B_(26)Tyr,B_(27)Thr,B_(28)Pro及B_(29)Lys谱峰的专一识别。由碱侧pH滴定,测得B_(16)Tyr,B_(26)Tyr的pK值分别为10.65及10.60。