Electrospray tandem mass spectrometry in the rapid identification of alpha-chain haemoglobin variants

The potential use of electrospray tandem mass spectrometry in the rapid characterisation of haemoglobin variants found in the Swedish population has been assessed. Analysis times of the order of 5 -10 min were routinely achieved, and identification of variants using mass spectrometry as the sole analytical technique was possible. However, additional information, readily available from isoelectric focusing experiments, made identification simpler and more secure. In the present communication we report on the identification of the alpha-chain variants, Hb Russ, Hb Le Lamentin and Hb Q-Iran. The identifications were confirmed by the use of nucleotide sequencing techniques.     

Isolation and preparative purification of microcystin variants

Preparative reversed-phase liquid chromatography was successfully used to purify two microcystins (microcystin LR and microcystin LA) from a cyanobacterial process waste. The separation protocol involved extraction of lyophilized cells by methanol, isolation and concentration by solid-phase extraction, and purification by reversed-phase HPLC. Milligram-level loading of microcystins was obtained on a solid-phase extraction cartridge packed with 0.5 g of C18 stationary phase. The separations were first carried out on an analytical column and then scaled-up to a preparative column. The microcystins were quantified by HPLC and enzyme-linked immunosorbent assay. A method to remove microcystins rapidly and economically from the cyanobacterial process waste is also described.     

Reversed-phase high-performance liquid chromatography of human haemoglobin chains

A reversed-phase high-performance liquid chromatographic method for the separation of human haemoglobin chains has been devised. Using a LiChrospher 100 CH-8/2 column and a ternary eluent (acetonitrile-methanol-0.155 M NaCl, pH 2.7) improved resolution was achieved between (delta beta) Lepore, beta A, beta S, alpha, G gamma and A gamma chains within a 60-min linear gradient. The A gamma T chain can also be separated by increasing the gradient time and decreasing the flow-rate. Silanophilic interactions play an important role in the retention mechanism, and NaCl addition was necessary in order to suppress adsorption on free silanols. Increasing the methanol concentration to 10% caused a slight increase in chain retention, probably owing to solvation of the stationary phase. The recovery was 82% and the reproducibility of retention times was as good as +/- 1.5%. Quantitation of chains is likely to be possible by peak area measurement. Owing to its sensitivity, the proposed method may be useful in the diagnosis of haemoglobinopathies and in the study of haemoglobin variants.     

Fructosylvaline. A simple model of the N-terminal residue of human haemoglobin A1c

The phenylhydrazone of N-[D-fructosyl-(1)]-L-valine (1-deoxy-L-valine-D-fructose) was synthesized. The hydrazone was shown to exist in open form in basic solution and in closed form in acidic solution. The findings have bearings upon the discussion of the reaction of human haemoglobin A1c with phenylhydrazine.     

Isolation of human serum immunoglobulins with a new salt-promoted adsorbent

In this work a highly acetylated-ethylenediamine-Novarose (HA-EDA-Novarose) gel was synthesized and used as a new agarose-based salt-promoted adsorption chromatography (SPAC) matrix to effectively isolate serum immunoglobulins without the need of denaturing conditions. Samples of human serum in 0.5 M Na2SO4, 10 mM 3-(N-morpholino)-propane-sulfonic acid (MOPS), pH 7.6 were applied to a chromatographic column packed with the SPAC gel. Immunoglobulins (Igs) with affinity for the HA-EDA ligands were specifically adsorbed to the matrix, non-bound serum proteins were readily removed by washing the column with the same feed solution buffer. Bound Igs were effectively and very gently eluted by simply removing the salt from the feed solution buffer. The elution buffer consisted thus of only 10 mM MOPS, at pH 7.6 and no salt. The salt-dependent adsorption capacity of this system was estimated to be 7.3 mg/ml with protein recovery of about 93%. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis analysis, radial immunodiffusion and enzyme-linked immunosorbent assays showed that immunoglobulins G, M and A (IgG, IgM and IgA) were the main components present in the elution fraction. The new SPAC adsorbent was used to purify Igs from human serum and IgG and IgA from non-pure commercially available Igs preparations in a very gentle single step.     

Isolation of acetic acid-tolerant Baker’s yeast variants in a turbidostat

A commercial baker’s yeast was subjected to selection in a continuous turbidostat cultivation with increasing concentration of acetic acid. The final acetic acid content in fresh medium was 0.6% or 0.8% v/v. Two of seven selected variants were stable over 15 sequential shake flask cultivations without selection pressure. After laboratory scale production of baker’s yeast, one of the variants also showed increased acetic acid tolerance in sour dough. The overall raising power (mL CO₂/h) in sour dough was improved 36%.     

Genetic studies of low-abundance human plasma proteins. X. Coagulation factor XIIIB variants in blacks

Human coagulation factor XIIIV (F XIIIB) demonstrates genetically determined-structural variation with three common and several rare alleles. Population genetics studies reveal enormous intra and interracial group variation. In the present study, using isoelectric focusing and immunoblotting, we have determined for the first time the polymorphic occurrence of F XIIIB allelic forms in a native African population, namely Nigerian Blacks. In addition, F XIIIB data have been extended to various US Black populations. The characteristic feature of the black gene pool is the relative high frequency of the F XIIIB*2 allele, the highest being in Nigerians (0.723). The F XIIIB*6 allele is present at a polymorphic level in both the US and Nigerian Blacks and appears to be a unique black allele marker. The present technique has demonstrated several new alleles designated: F XIIIB*18, FXIIIB*22, F XIIIB*23 and F XIIIB*24. Among these new alleles the F XIIIB*23 exists at polymorphic level in both the US and Nigerian Blacks and is another unique Black allele marker of potential significance in population genetics studies.     

Preparation of Tyr-C-peptide from genetically altered human insulin precursor

C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity.¹²⁵I labeled TyrC-peptide is indispensable in C-peptide RIA kit. Herein we discuss a way of obtaining recombinant Tyr-C-peptide. Arg32Tyr human proinsulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed inEscherichia coli. Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion. Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis.     

Cysteine adducts of human haemoglobin measured by isoelectric focusing in polyacrylamide gels with a non-linear pH gradient

The in vitro formation of adducts from human haemoglobin formed by alkylation with methyl-methanesulphonate, dimethyl sulphate and iodoacetamide was determined with isoelectric focusing in ultra-thin polyacrylamide gels with a non-linear pH gradient. The most important adduct seen in the gels was identified as HbA alkylated at the beta-93 cysteine. Influences of the chemical nature of the alkylating agents and of the biological environment are discussed. The method is suggested to be applicable to monitoring the biological effects of low, long-term exposure to mixtures of alkylating agents or of exposure to unknown compounds.     

Separation of human alloalbumin variants by isoelectric focusing

A technique for the separation of human alloalbumin variants by means of isoelectric focusing in the presence of 8M urea and 60 mM L-serine is described. The potential usefulness of this technique in the detection and classification of genetic heterogeneity at the albumin locus is demonstrated by the differentiation of three human alloalbumin variants of European origin.     

Automated quantitative microcolumn chromatography of haemoglobin A2.

Automated cation exchange microcolumn chromatography of haemoglobins has been modified for the analysis of haemoglobin A2. It provides the quantitative data of sufficient precision and specificity for the investigation of potential heterozygotes for beta-thalassaemia. Results have been compared with an established method of electrophoresis followed by densitometry of the eluted bands.     

Increased-valence formulae and the bonding of oxygen to haemoglobin

The molecular orbital configuration for the ground-state of O₂ generates the valence formula . “Increased-valence” formulae for triatomic and polyatomic molecules have been developed recently. In them, more electrons participate in bonding than is possible for the familiar valence-bond formulae. Using oxyhaemoglobin as an example of an oxygen carrier, various increased-valence formulae are generated for the FeO₂ groups. In the low-energy formulae, the iron is bonded to the above valence formula for O₂. Therefore, in contrast to the bonding schemes of Pauling and Coryell, Griffith and Weiss, little reorganization of O₂ need occur on formation of HbO₂. This conclusion is independent of the mode of co-ordination of iron to the O₂.     

Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography

In this report, we have demonstrated the isolation and enrichment of charge variants of a monoclonal antibody IgG1 using cation exchange displacement chromatography. We successfully achieved the separation of acidic, main and basic charge variants with high recovery (>70%) and purity (>90%) by using a commercially available stationary phase in conjunction with a commercially available displacer. In addition, we have isolated and enriched a trace methionine-oxidized variant of the monoclonal antibody allowing a secondary means of identification of this variant while providing sufficient enrichment for further analysis, stability tests and potency determination. Further characterization of the displacement trains by SEC indicate the possibility of enrichment of high and low molecular weight species. Glycan analysis of the displacement fractions indicates minimal variation in glycan distribution patterns among a wide spectrum of charge variants. These results provide a case study demonstrating the utility of cation exchange displacement chromatography as a viable approach to isolate and enrich antibody charge variants for enhanced molecular characterization.     

High-performance anion-exchange chromatographic study of desialylated human alpha 1-acid glycoprotein variants. Development of a fractionation method for the protein slow variants

The three main desialylated variants (F1, S and A) of human alpha 1-acid glycoprotein (AAG), a serum acute-phase reactant, were analysed by high-performance anion-exchange chromatography in order to determine their optimum separation conditions. The analysis consisted of three steps, as follows: (1) A desialylated commercial AAG was separated into one "fast"- and one "slow"-migrating fraction by preparative isoelectrofocusing. The "fast" and "slow" fractions were shown to contain the F1 variant and a mixture of the S and A variants, respectively. (2) The pH titration curves of these two fractions were then measured by strong anion-exchange chromatography with several buffer systems of increasing pH. From the data obtained, it was not possible to select the optimum conditions to separate the "fast" variant F1 from the "slow" variants A and S. However, the S and A variants were shown to ionize very differently. (3) The specific fractionation of the S and A variants was therefore carried out by anion-exchange chromatography under operating conditions based on the data obtained from the study of their pH titration curves. This was performed both with the "slow"-migrating fraction obtained by preparative isoelectrofocusing of commercial AAG and with an AAG (containing only variants S and A) purified from an individual serum on immobilized Cibacron Blue F3G-A. Identification of the fractionated proteins was achieved by analytical isoelectrofocusing.     

Isolation and evaluation of cytogenetic effect of Brahmi saponins on cultured human lymphocytes exposed in vitro

Major saponins of Brahmi (Bacopa monniera, Fam: Scrophulariaceae) – bacosides A and B – were isolated from the total methanol extract and characterised based on melting point, TLC, IR, ¹H NMR and ¹³C NMR. They were evaluated for their in vitro cytogenetic effects on human peripheral blood lymphocytes by chromosomal aberration (CA) assay and sister chromatid exchange (SCE) assay. The frequency of chromatid type aberrations and reciprocal interchanges between sister chromatids in the treated cells was scored in comparison to the untreated control. At 30 μg/mL dose, bacoside A showed a statistically significant increase in the frequency of both CA and SCE and bacoside B showed an increase only in SCE. Our report of the genotoxicity of the saponins is significant in view of the reports of anticancer activity of Brahmi extracts.