Kiyohito Shimura

Estimation of the Deamidation Rates of Major Deamidation Sites in a Fab Fragment of Mouse IgG1-κ by Capillary Isoelectric Focusing of Mutated Fab Fragments

The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modification. The identification of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such analysis becomes difficult and sometimes impossible. In this report, a quantitative method for estimating the deamidation rate of a specific amide in a protein is presented without using peptide mapping. Five Asn residues of a recombinant fragment antigen binding (rFab) (mouse IgG1, κ) were mutated to a serine (Ser) residue, one by one, through site-directed mutagenesis, and the single-residue deamidation rates of the original rFab and the mutants were determined using capillary isoelectric focusing. The difference of the rate between the original rFab and the mutant was assumed to be equal to the deamidation rate of the specific Asn residue, which had been mutated. Among five mutants established, three major deamidation sites-H chain Asn135, L chain Asn157, and L chain Asn161, using the Kabat numbering system-were identified, accounting for 66%, 29%, and 7% of the single-residue deamidation of the original rFab, respectively. Although the former two have been known by peptide mapping, the last one, which resides on the same tryptic peptide that carries one of the former two, previously has not been identified. For the first time, the deamidation rate constants of the three sites were estimated to be 10.5 × 10(-3) h(-1), 4.6 × 10(-3) h(-1), and 1.1 × 10(-3) h(-1) in 0.1 M phosphate buffer, pH 7.5 at 37 °C, respectively, with corresponding half-life of 2.8 days, 6.3 days, and 27 days. The method should be applicable to any recombinant proteins.

Affinophoresis: selective electrophoretic separation of proteins using specific carriers.

Publisher Summary Affinophoresis is an electrophoretic procedure that uses a specially designed carrier molecule aimed at modifying the mobility of a target substance. The power of the method as an analytical procedure is well demonstrated by the linkage of two-dimensional affinophoresis, with immunoblotting or enzyme activity measurements. Other modes of two-dimensional application, e.g, combination with isoelectric focusing, should also be effective. The same affinophoretic principle can be applied, by using polyionic biomolecules, such as polynucleotides and mucopolysaccharides, as natural equivalents for affinophores. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and electrokinetic chromatography can be considered analogous to affinophoresis, although they do not depend on biospecific affinity. Affinophores, bearing only ligands of low molecular weight, are described in this chapter. Various binding proteins, such as antibodies and lectins, can be used as ligands as well. Possible ligands are also not limited to biomolecules. A variety of interacting systems that have been utilized in affinity binding, e.g., protein-dye, sugar-boronate chelating group- metalloprotein, and hydrophobic interaction, are the candidates. It would be useful if neutral complex carbohydrates could be separated according to their structure, for example, by using affinophores, bearing specially oriented boronate groups. Although preparation of a special affinophore for each target may appear troublesome, a more general affinophore can be prepared, by linking, with the avidin-biotin system.

High-performance affinity chromatography of a chick lectin on an adsorbent based on hydrophilic polymer gel

A mouse monoclonal antibody (SIA4-5) which reacts with a chick 14K lectin (C14K) was covalently attached to a new support for high-performance affinity chromatography, TSKgel Tresyl-5PW, which is a preactivated, polymer-based particle. The immobilized antibody (SIA4-5-5PW) thus prepared proved to be useful in measuring not only the molecular properties of C14K but also specific interactions of C14K with SIA4-5 and hapten sugars. The C14K preparation was fractionated according to the oligomeric structure and with slight differences in affinity to SIA4-5 although the former was homogeneous in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Application of the method for quantitative analytical purposes was successful.

Affinity probe capillary electrophoresis of insulin using a fluorescence-labeled recombinant Fab as an affinity probe.

Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence‐labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti‐insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol‐reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab‐gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral‐polymer coated fused silica capillary (50 μm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser‐induced fluorescence under focusing conditions. The insulin‐AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first‐order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.

Analysis of Lectin–Carbohydrate Interactions by Capillary Affinophoresis

Publisher Summary Lectin–carbohydrate interactions represent a typical area where affinity constants have been determined by observing the migration of lectins in gel matrices to which carbohydrates are immobilized. The migration of lectins is diminished by the interaction with the immobilized carbohydrates in these applications. This chapter describes a different approach, in which carbohydrate ligands are attached to soluble ionic polymers. In this case, the electrophoretic migration of lectins is enhanced by the interactions. Ligand–ionic polymer conjugate is referred to as an “affinophore” and electrophoresis, using the affinophore, as “affinophoresis.” Typically, polyliganded affinophores are prepared by coupling p -aminophenyl glycosides to an anionic polymer, succinylpolylysine, at a glycoside—succinyllysine ratio of about 10%. Capillary electrophoresis has many characteristics that recommend it in the analysis of molecular interactions, that is, the ability to analyze interactions in free solutions, a short analysis time, precise temperature control, and a small sample size. Although polyliganded affinophores are also effective in detecting lectin–carbohydrate interactions in a capillary, they are not suitable for the determination of affinity constants because of the multivalency of most lectins. Monoliganded affinophores were developed to solve this problem, and affinity constants between divalent lectins and carbohydrates were determined by capillary affinophoresis performed in a competitive manner.

Capillary isoelectric focusing after sample enrichment with immunoaffinity chromatography in a single capillary

For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution. An anode solution, an acid, was then introduced to fill only the IAC column segment. Focusing was started with a pressure that balances with the electroosmotic flow produced in the acidified IAC column. Fluorescence-labeled recombinant Fab with an E-tag spiked at 16 pM to 10u2009nM in 50% serum was separated and detected with high precision. The coupling principle allows rapid and high-resolution IEF analysis of a protein in a biological sample without any loss of the immunoaffinity captured protein.

Quantitative evaluation of lectin-reactive glycoforms of α1-acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection

α1‐Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein‐labeled AGP using lectins with the aid of laser‐induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A‐reactive or Aleuria aurantia lectin‐reactive AGP. Labeled AGP was applied at the anodic end of a fused‐silica capillary (50u2009μm id, 360u2009μm od, 27u2009cm long) coated with linear polyacryloyl‐β‐alanyl‐β‐alanine, and electrophoresis was carried out for about 10u2009min in 60u2009mM 3‐morpholinopropane‐1‐sulfonic acid‐NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin‐reactive glycoform peaks from lectin‐non‐reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin‐reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5u2009mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro‐purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies.

Capillary Isoelectric Focusing

Abstract In isoelectric focusing (IEF), ampholytes like proteins are concentrated at different positions in a pH gradient. When their isoelectric points (pI) are different, they are separated. Capillary isoelectric focusing (CIEF) is a rapid, high-resolution separation method for ampholytes at a microscale. The chapter explains the basic concept of the natural pH gradient formed with a carrier ampholyte and then focuses on separation and detection of proteins in the capillary format. The essential components of CIEF, i.e., capillary coatings, spacers, pI markers, mobilization of focused ampholytes, and whole-column detection, are concisely discussed.

Cesium reversibly suppresses HeLa cell proliferation by inhibiting cellular metabolism

The aim of the present study was to investigate the influence of Cs+ on cultured human cells. We find that HeLa cell growth is suppressed by the addition of 10 mm CsCl into the culture media. In the Cs+‐treated cells, the intracellular Cs+ and K+ concentrations are increased and decreased, respectively. This leads to a decrease in activity of the glycolytic enzyme pyruvate kinase, which uses K+ as a cofactor. Cs+‐treated cells show an intracellular pH shift towards alkalization. Based on these results, CsCl presumably suppresses HeLa cell proliferation by inducing an intracellular cation imbalance that affects cell metabolism. Our findings may have implications for the use of Cs+ in cancer therapy.