Technical University Bergakademie Freiberg, Institute of Analytical Chemistry, Leipziger Straße 29, D-09599 Freiberg, Germany.
J Chromatogr A. 2011 Jan 14;1218(2):280-5. doi: 10.1016/j.chroma.2010.11.031. Epub 2010 Nov 23.
A separation method for a mixture of eight sulfur-containing peptides and proteins characterized by a wide molar mass (1-18.4 kDa) and pI range (4.5-10.7) was developed onto a monolithic phenyl phase. Based on the first optimization steps that revealed an increase of the acetonitrile content to 45 vol.% as sufficient for the elution of all biomolecules and the addition of the ion pairing reagent trichloroacetic acid (TCA) as preferable over the eluent additives formic acid or ammonium acetate buffer, the critical variables TCA concentration, gradient time, and eluent flow rate were optimized using a Box-Behnken experimental design. To achieve optimum values for separation factors of all peak pairs, a TCA content of 0.025% (m/v), a gradient time of 10 min, and a flow rate of 3.5 mL min(-1) were selected. Arsenic binding studies were undertaken under conditions optimized with respect to the crucial separation factor of the nonapeptides vasotocin (Vtc) and vasopressin (Vpr) in a shortened gradient time of 7.5 min. A complete separation of phenylarsenic-substituted and unmodified forms of these peptides allowed the calculation of both consumptions and apparent equilibrium constants K from HPLC-UV peak areas. The nonapeptide consumptions by the reaction with phenylarsine oxide (PAO) increased from 7% up to 100% in dependence on the molar ratio of the reaction components. Due to an enhanced UV absorption of the phenylarsenic-substituted biomolecules, the calculation of apparent equilibrium constants led to increasing K values with rising PAO molarities from 9.6×10(5) to 1.2×10(8) in case of Vtc and from 2.2×10(6) to 1.4×10(9) in case of Vpr. For α-lactalbumin, a consumption of 59.2±6.1% by the reaction with molar excesses of PAO varying from 1.4 to 21 can be derived from the chromatograms. The quantitative evaluation of the reaction of the small protein aprotinin with PAO was hindered by a pronounced peak broadening that occurred after reduction of the disulfide bridges.
一种用于分离八种含硫肽和蛋白质混合物的方法，其特点是摩尔质量（1-18.4 kDa）和等电点（4.5-10.7）范围较宽，已被开发到整体苯基相上。基于首次优化步骤，发现增加乙腈含量至 45%体积比足以洗脱所有生物分子，并且添加离子对试剂三氯乙酸（TCA）比洗脱剂添加剂甲酸或乙酸铵缓冲液更可取，然后使用 Box-Behnken 实验设计优化了 TCA 浓度、梯度时间和洗脱液流速等关键变量。为了实现所有峰对分离因子的最佳值，选择了 TCA 含量为 0.025%（m/v）、梯度时间为 10 min 和流速为 3.5 mL min(-1)。在缩短梯度时间为 7.5 min 的情况下，根据非肽血管加压素（Vtc）和血管升压素（Vpr）的关键分离因子，进行了砷结合研究。完整分离这些肽的苯砷取代形式和未修饰形式，允许根据 HPLC-UV 峰面积计算两种消耗和表观平衡常数 K。由于苯胂化生物分子的紫外吸收增强，反应中与苯胂化氧（PAO）的非肽消耗从 7%增加到 100%，这取决于反应成分的摩尔比。由于 Vtc 的 K 值从 9.6×10(5)增加到 1.2×10(8)，Vpr 的 K 值从 2.2×10(6)增加到 1.4×10(9)，因此，随着 PAO 摩尔浓度的增加，表观平衡常数增加。对于α-乳白蛋白，可以从色谱图中得出，当 PAO 的摩尔过量从 1.4 到 21 时，反应消耗 59.2±6.1%。由于二硫键还原后出现明显的峰展宽，因此对小蛋白 aprotinin 与 PAO 的反应进行定量评估受到阻碍。
