We have plotted electropherograms in capillary zone electrophoresis (CZE) as a function of the quantity of electric charge (Q) in order to eliminate the dependency of the analyte peak areas, as well as that of the migration times, upon both the capillary temperature and the applied voltage. The procedure is based on an idea of a migration index (MI) and an adjusted migration index (AMI) which were originally proposed by Lee and Yeung. The value of Q is measured accurately and calculated easily because it is given by a product of the electrophoretic current and the migration times, where the index MI is derived by dividing the value of Q by the effective volume of the capillary. By calculating the CZE peak area from the newly plotted electropherogram, improvement in precision in quantitative analysis is expected. Concerning AMI, careful treatment is required in its application to analyte peaks whose migration time is close to that of the neutral marker. Experimental data and discussions concerning the migration indices are presented.

This paper describes the suppression and control of the racemization ratio (D or L/D+L) of phenylthiohydantoin (PTH) amino acids in the Edman sequencing method. Most of the racemization occurs in the cyclization/cleavage step. Although optimization of partial racemization using a mixture of TFA and boron trifluoride (BF3)-ethyl ether complex, which is effective in suppressing racemization in the cyclization/cleavage reaction. The partial racemization in PTH derivatization is often useful for DL differentiation, because a minor L- or D-peak produced by racemization can be used as an internal standard in CE. Using the partial racemization method with mixed acids as a cyclization/cleavage reagent, the sequence determination of [D-Ala2]-methionine enkephalin, with DL differentiation, was achieved on a sequencer.

A comparative study of the reproducibility in migration times between constant-current (CC) and a constant-voltage (CV) mode of operation in capillary zone electrophoresis was carried out. We found that the CC mode gave better reproducibility than the CV mode in both successive injections (5 or 6 times) and day-to-day analyses (16 measurements throughout 4 days).

We have constructed a simple, low-cost, time-resolved fluorescence detector (TRFD) for analysis of amino compounds with high-performance liquid chromatography using europium (Eu3+) chelates, which consists of a dc-operated xenon lamp, two synchronized mechanical choppers, and spectroscopic filters. Amino compounds derivatized by isothiocyanobenzyl EDTA with Eu3+ chelate are mixed with an enhancer solution postcolumn and detected by the TRFD. Taking advantage of the long fluorescence lifetime of the Eu3+ chelates, high selectivity against background fluorecein is achieved. To demonstrate the usefulness of the TRFD, fundamental performance test comparison with the conventional fluorescence detector was carried out. Details of the system are also described.

Indirect time-resolved fluorescence (ITRF) detection of non-fluorescent phenylthiohydantoin (PTH)-Ala and fluorescent dansyl (DNS)-Ala separated by high-performance liquid chromatography (HPLC) was carried out. Addition of europium (Eu3+) chelates to the eluent makes it possible to detect both non-fluorescent and fluorescent compounds simultaneously. This is because the fluorescence lifetime of Eu3+ chelates is extremely long, whereas that of normal fluorescent compounds is short. The HPLC conditions with the Eu3+ chelate system were studied.

An advanced method is described for the complete separation of phenylthiohydantoin (PTH)-DL-amino acids for protein sequencing. Optical resolution of all standard PTH-DL-amino acids was successfully developed using some chiral selectors, although the resolution of only PTH-DL-His and Lys could not be confirmed, due to low reproducibility and the presence of impurities.In addition, mixed chiral selectors for making a single electrolyte with the ability to optically resolve all standard PTH-DL-amino acids were investigated. Using the only resulting electrolyte, sequence determination of [D-Ala(2)]-methionine enkephalin, with or differentiation, was performed.

This is an initial report to propose a protein sequence analysis system with DL differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE system. After fractionation of phenylthiohydantoin (PTH)-amino acids using a protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, beta-escin and others. As a model peptide, [D-Ala(2)]-methionine enkephalin (L-Tyr-D-Ala-Gly-L-Phe-L-Met), was used and the sequence with DL differentiation was determined, with the exception of the fourth amino acid, L-Phe, using our proposed system.

未処理フューズドシリカキャピラリーを用いたキャピラリー電気泳動法によって, 11種のモデルタンパク質 (MW 6～75Kd: pI4.5～11.2) 及びヒト血清タンパク質の分離分析を試みた. 電解液, 添加剤, キャピラリー洗浄法, 試料注入法について, タンパク質の移動時間,ピーク面積の再現性を指標として検討した. 電解液として100mMホウ酸ナトリウム (pH10.0), 洗浄液として0.1Mリン酸水溶液 (pH2.5), 泳動ごとに洗浄液と電解液を交互に5分間送液してキャピラリーの初期化を行い, 再現性の良い分離分析が達成できた. 使用したモデルタンパク質は, チトクロムcを除き, 等電点と相関する移動時間を示した. ヒト血清タンパク質は, 8分以内で分離分析が可能であった. さらに改善すべき点はあるが, 未処理フューズドシリカキャピラリーを用いた電気泳動法は, 微量, 高分離, 迅速, 簡便なタンパク質分析手法として有用であることを確認した.

健常人および患者血清タンパク質を未処理フューズドシリカキャピラリーを用いたキャピラリー電気泳動法で分離分析した. 血清タンパク質をアルブミン, α1-グロブリン, α2-グロブリン, β-グロブリン, γ-グロブリンの各分画として, それらの成分比をセルロースアセテート膜を担体とした電気泳動法より得られる結果と比較した. CEにおいて, α1分画が多めに (CA法における相対成分比の平均値2.3に対してCE法は4.6) 検出されたことを除けば, よく一致する値を得た.

An immersed flowcell has been developed for fluorescence detection in capillary electrophoresis (CE) by modifying an ordinary HPLC spectrofluorimetric detector flowcell. The minimum detectability obtained for riboflavine was about 4 fmol. The use of the flowcell to measure a fluorescence spectrum by the stopped-flow method of shutting down the DC high voltage during a wavelength scan, has also been achieved successfully.

In this study. using a single solvent system (30 mM sodium phosphate buffer at four different pH values: 2.0, 5.0, 7.0 and 10.0). we have investigated the migration behaviors for not only small molecules (more than 35 peptides varying in size from 2 to 42 amino acid residues and varying in charge from -1.44 to 6.93) but also large ones (more than 20 proteins varying in molecular weight from 5 kDa to 480 kDa and varying in isoelectric point from 4.1 to 11 0) Though the size (radius) of a molecule has been difficult to evaluate, it showeg best linear correlation at pH 2.0 (r 0.981). with respect to Mw (2/3) as radius of a peptide. Furthermore. it was observed that a good correlation between observed and gatculated mobility was only up to approximately 20 kDa. using Mw (273) for R and Z (calculated from the pKa of amino acid residues) at pH 2.0.

High-performance liquid chromatography combined with circular dichroism spectrophotometry (HPLC-CD) for the separation and conformational analysis of proteins is described. The HPLC-CD measurement of proteins was performed easily and reproducibly with a gradient elution method by using a micro-flow cell unit. Reversed-phase chromatography and hydroxyapatite chromatography of proteins with this system were demonstrated. In addition, the influence of salts, surfactants and the interaction of packing materials on the secondary structure of proteins during HPLC was investigated and the α-helix content of protein was calculated by using a secondary structure estimation program.

Basic studies of the combined system of a high performance liquid chromatograph (HPLC) and a circular dichroism (CD) spectrometer for separation and analysis of proteins are described. The HPLC-CD measurement of standard protein mixture was easily carried out by using a micro flow-cell device with a beam condenser and with a thin cell of a 1 mm-optical path. The effluent was firstly monitored at 280 nm by using an UV detector and subsequently monitored at 220 nm by using a CD spectrometer. The CD spectrum at each chromatographic peak by CD was measured in the wavelength region of 250–195 nm by a stopped flow method.

The degradation process of calmodulin with trypsin in the presence or absence of calcium ions was reinvestigated by the analysis on HPLC. All tryptic peptides and intermediate peptides were mapped on this system. On the basis of these results, the strategy of the preparation of various calmodulin fragments was devised. Twelve peptides which contain calcium binding sites were obtained.