Hansen P, Andersson L, Lindeberg G
School of Biochemistry, La Trobe University, Bundoora, Australia.
J Chromatogr A. 1996 Feb 2;723(1):51-9. doi: 10.1016/0021-9673(95)00806-3.
Peptides containing a cysteine residue but lacking histidine and tryptophan were synthesised by the solid-phase method. Their retention behaviour on Cu2+ - and Ni2+ -loaded immobilised metal ion affinity chromatography (IMAC) supports at pH 5-11 was studied and compared with that observed for the corresponding compounds without the free alpha-amino group and/or the thiol function. Unexpectedly, it was found that neither a cysteine side-chain nor a cysteine disulphide affects the retention of the peptides. A free alpha-amino group is required for binding; no retention is observed in its absence. At pH 9 substantial amounts of metal ions were transferred from the chromatographic support to an alpha-amino-protected cysteine-containing peptide. However, at pH 7 no such transfer occurred. Therefore, the lack of retention observed for peptides with a blocked alpha-amino function over the entire pH range is not solely caused by metal ion scavenging by the thiol group. Partial dimerisation may occur upon chromatography; the dimers formed are retained strongly due to the presence of two free alpha-amino groups. It seems that IMAC on a Cu2+ - or Ni2+ -loaded support can be used for the purification of cysteine-containing peptides synthesised by the solid-phase method. Inclusion of a capping protocol in the synthesis ensures that a free alpha-amino group, which can be used as an affinity handle, will be present only on the target peptide.
通过固相法合成了含有半胱氨酸残基但缺乏组氨酸和色氨酸的肽。研究了它们在pH 5 - 11条件下在负载Cu2+和Ni2+的固定化金属离子亲和色谱（IMAC）支持物上的保留行为，并与相应的没有游离α-氨基和/或硫醇功能的化合物的保留行为进行了比较。出乎意料的是，发现半胱氨酸侧链和半胱氨酸二硫键均不影响肽的保留。结合需要一个游离的α-氨基；没有它则观察不到保留。在pH 9时，大量金属离子从色谱支持物转移到α-氨基保护的含半胱氨酸的肽上。然而，在pH 7时没有发生这种转移。因此，在整个pH范围内，具有封闭α-氨基功能的肽缺乏保留并非仅仅是由于硫醇基团清除金属离子所致。色谱过程中可能会发生部分二聚化；由于存在两个游离的α-氨基，形成的二聚体被强烈保留。似乎负载Cu2+或Ni2+的支持物上的IMAC可用于纯化通过固相法合成的含半胱氨酸的肽。在合成中加入封端方案可确保仅在目标肽上存在可作为亲和手柄的游离α-氨基。
