Yasuyuki Kurosu

Direct Combination of a High Performance Liquid Chromatograph and a Circular Dichroism Spectrometer for Separation and Structural Analysis of Proteins

Abstract 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.

Comparison of the reproducibility in migration times between a constant-current and a constant-voltage mode of operation in capillary zone electrophoresis

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).

Analysis of proteins by high-performance liquid chromatography with circular dichroism spectrophotometric detection

Abstract 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.

Electropherograms in capillary zone electrophoresis plotted as a function of the quantity of electric charge

Abstract 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.

Optical resolution of phenylthiohydantoin-amino acids by capillary electrophoresis for protein sequencing

Abstract 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 -Ala2]-methionine enkephalin, with dl differentiation, was performed.

Time-resolved high-performance liquid chromatography fluorescence detector using a nanosecond pulsed light source for detecting lanthanide-chelated compounds

We have constructed a time-resolved fluorescence detection (TRFD) system for the analysis of amino compounds with high-performance liquid chromatography (HPLC) using lanthanide ion chelates. In order to carry out time-resolved measurements, we have employed a nanosecond pulsed xenon-arc lamp as an excitation light source. Amino compounds derivatized by isothiocyanobenzyl-EDTA (IEDTA) with the lanthanide chelate are mixed with an enhancer solution in a post-column manner and detected by TRFD. Taking advantage of a property of the long fluorescence lifetime of the lanthanide chelates, high selectivity against background fluorescence was achieved. In order to demonstrate the usefulness of TRFD, fundamental performance tests were carried out. Details of the system are also described.

Optical resolution of phenylthiohydantoin-amino acids and identification of phenylthiohydantoin-d-amino acid residue of [d-Ala2]-methionine enkephalin by capillary electrophoresis

Abstract We propose a system of protein sequence analysis with dl differentiation using capillary electrophoresis (CE). This system consists of a protein sequencer and a CE. After fractionation of phenylthiohydantoin (PTH)-amino acids from the protein sequencer, optical resolution for each PTH-amino acid is performed by CE using some chiral selectors such as digitonin, o -trimethyl-β-cyclodextrin (TM-β-CD) and others. In addition, optical resolution of all standard PTH- dl -amino acids including PTH- dl -carboxymethyl-Cys (CM-Cys) and cysteic acid (CYA) except for PTH- dl -Lys was successfully developed. The resolution of PTH- dl -Lys could not be reconfirmed due to low reproducibility and the impurities. 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 completely determined.

Identification of Chirality of Phenylthiohydantoin-D-Amino Acid Residue of [D-ala2]-Metthionine Enkephalin by Capillary Electrophoresis: Suppression and Control of Racemization Ratio in the Edman Sequencing Method

Abstract 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.

Indirect time-resolved fluorescence detection of both non-fluorescent and fluorescent compounds separated by high-performance liquid chromatography

Abstract 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 (Eu 3+ ) chelates to the eluent makes it possible to detect both non-fluorescent and fluorescent compounds simultaneously. This is because the fluorescence lifetime of Eu 3+ chelates is extremely long, whereas that of normal fluorescent compounds is short. The HPLC conditions with the Eu 3+ chelate system were studied.