Production of functional human fetal hemoglobin in Nicotiana benthamiana for development of hemoglobin-based oxygen carriers.

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.

Comparison of fast protein liquid chromatography (FPLC) with HPLC, electrophoresis & microcolumn chromatography techniques for the diagnosis of beta-thalassaemia.

BACKGROUND & OBJECTIVE: beta-thalassaemia is a genetic disorder and an important health problem around the world. Quantitative haemoglobin A(2) (HbA(2)) levels are used for the diagnosis of beta-thalassaemia. The conventional methods are high performance liquid chromatography (HPLC), electrophoresis, and microcolumn chromatography techniques. We established a fast protein liquid chromatography (FPLC) method, to measure quantitatively of HbA(2) levels, and compared its efficacy with conventional methods. METHODS: The FPLC method, using a DEAE Sepharose, Hi Trap anion-exchange column chromatography technique was set up for HbA(2) measurement. In this study, 220 blood samples were screened for haemoglobin type by FPLC technique and also using HPLC, microcolumn chromatography and electrophoresis. RESULTS: The FPLC results were highly correlated (r = 0.985, P<0.001) with those of HPLC for quantification of HbA(2) as well as cellulose acetate electrophoresis (r = 0.977) and microcolumn chromatography (r = 0.980). The FPLC method showed 100 per cent sensitivity and specificity, positive and negative predictive value for beta-thalassaemia diagnosis. In addition, the FPLC method was simple, rapid, low cost and reproducible. The HbA(2)/E range of FPLC for beta-thalassaemia was 6-10 per cent, HbE trait was 10-40 per cent, beta-thalassaemia/HbE was 40-60 per cent and homozygous HbE was more than 60 per cent. INTERPRETATION & CONCLUSION: Our findings suggested that FPLC method could be used as a cost-effective method for routine beta-thalassaemia diagnosis.

Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing.

We developed two capillary isoelectric focusing (CIEF) assays, in narrow pH gradients, with the aim of routinely separating and quantitating normal and abnormal hemoglobins (Hbs): a one-step CIEF assay where residual electroosmotic flow mobilizes the proteins during focalization, and a two-step CIEF assay where focused Hbs are mobilized by low pressure by maintaining high-voltage. The resolution of 0.10 pH unit obtained with the one-step assay allowed the separation of the Hbs A, F, S and C; but Hb A2, which represents about 2-3% of whole Hb, could not be quantitated. The better resolution of 0.02 pH unit obtained with the two-step assay allowed the separation of some Hb variants of very close isoelectric points. The reproducibility of retention times was satisfactory (C.V.<5%). Moreover, in this configuration quantitation of Hb A2, Hb F and Hb S led to a standard deviation of less than 5%, allowing the diagnosis of thalassemias. The one-step assay could be useful only for the detection of abnormal variants, while the two-step assay could be applied to the routine analysis of Hbs, with quantitation of minor fractions and presumptive identification of variants.

Isoelectric focusing in immobilized pH gradients: recent analytical and preparative developments.

Isoelectric focusing in immobilized pH gradients (IPG), covering both analytical and preparative aspects, is here reviewed. An extensive introduction covers the development of the technique from its inception in 1982 to present day methodology, with particular emphasis on the development of computer programs able to calculate and optimize linear and nonlinear pH gradients, spanning as much as 9 pH units, from a mixture of as many as 10 different buffering ions and titrants. The unique resolving power of IPGs is illustrated with the resolution of fetal globin chains differing by an Ala/Gly substitution in residue 75, this bringing about a minute difference in pI value of only 0.001 pH units. IPG runs, performed under denaturing conditions, allow an excellent correlation between experimental and theoretical protein pIs, to the extent that outliers were found to be polypeptide chains which had undergone post-synthetic modifications. The IPG methodology allows easy interfacing with mass spectrometry, due to the fact that proteins eluted from an IPG gel are isoionic as well as isoelectric, and thus are not contaminated by any buffer ion. The review ends with an excursus on preparative aspects of IPGs: a novel apparatus, based on the principle of isoelectric, buffering membranes, allows pilot-scale purification of r-DNA proteins to extreme purity, with recovery in a liquid vein. Isoelectric membranes have a selectivity based on a continuous titration process, and thus act as isoelectric traps for individual protein species. This same preparative apparatus can be used as a novel immobilized enzyme reactor, with superior performance compared to conventional types of reactors.

Identification of five embryonic hemoglobins of rat and ontogeny of their constituent globins during fetal development.

Hemoglobins of rats switch from an embryonic to an adult type during fetal development. However, very little is known about the structures and molecular species of hemoglobins occurring in the fetal life of rats. In the present study we isolated five embryonic hemoglobins, designated E1, E2, E3, E4, and E5, from the blood of rat fetuses on day 14 of gestation by ion exchange chromatography. Reverse-phase high performance liquid chromatography revealed that these hemoglobins each consist of two kinds of globins: E1(11 alpha:epsilon 1), E2(1 alpha: epsilon 1), E3(zetta: epsilon 1), E4(1 alpha: epsilon 3), and E5(zetta: epsilon 3), respectively. The complete amino acid sequences of the zetta, epsilon 1, and epsilon 3 globins were determined. The zetta globin showed characteristic features common in alpha-type embryonic globins of known species in that the N-terminus is blocked and the amino acid at position 38 is Gln. epsilon 1 and epsilon 3 are beta-type embryonic globins, sharing 73.7% amino acid homology. Interestingly, they are more similar to the corresponding mouse beta-type embryonic globins, y and z, respectively, than to each other, implying that these globins have evolved orthologously from common ancestral proteins. It was also shown that the zetta, epsilon 1, and epsilon 3 globins are almost completely replaced by the adult type alpha and beta globins in the blood of rat fetuses by day 18 of gestation.

Separation of hemoglobin variants in single human erythrocytes by capillary electrophoresis with laser-induced native fluorescence detection.

Single human red blood cells, in which the hemoglobin (Hb) molecules exist in their native, tetrameric states, were analyzed. Upon injection and lysis of a cell, the tetramers were dissociated on-column into their respective polypeptide chains, separated, and detected by laser-induced native fluorescence detection with 275-nm excitation. This technique was applied to the determination of hemoglobin variants as found in adult (normal and elevated Hb A1) and fetal erythrocytes. Normal adult cells contained 9.6% and 4.8% glycated beta- and alpha-chains, respectively. Cells with elevated Hb A1 gave 30% and 12%, respectively. The amounts of glycated Hb and total Hb in a given cell were found to be uncorrelated.

Fractionation of carrier ampholytes in multicompartment electrolyzers with isoelectric membranes.

Multicompartment electrolyzers with isoelectric membranes can be successfully utilized for the preparation of carrier ampholytes of narrow pH ranges from commercially available wider pH intervals. An example is given on the preparation of a 0.6 pH unit (pH 6.7-7.3) range from a standard pH 6-8 interval. A 10% Ampholine solution is focused in an electrolyzer equipped with the following isoelectric membranes: pI 6.0, 6.7, 7.3 and 8.0. The narrow pH 6.7-7.3 cut can be efficiently utilized for base-line separation and quantitation of hemoglobin A from its glycated form, Hb A1c. This analysis is important for the screening and follow up of diabetic patients. The advantage of multicompartment electrolyzers are: (i) precision in the preparation of narrow pH cuts, due to the presence of membranes with defined pI values; (ii) ability to perform in both small- and large-scale operations and (iii) absence of contaminants leaching from granulated supports, as typical of some previous techniques.

Functional alterations in adult and fetal hemoglobin Sassari Asp-alpha 126(H9)-->His. The role of alpha 1 alpha 2 contact.

The effects of pH, organic phosphates (2,3-diphosphoglycerate), and temperature on the functional properties of both adult and fetal hemoglobin Sassari alpha (Asp-126-->His) have been studied. The functional properties of the adult variant are characterized by the following: (i) an oxygen affinity higher than that of normal HbA in all the experimental conditions used; (ii) a dramatic reduction of homotropic interactions (n50 very close to unity); and (iii) a significant decrease of the effect of 2,3-diphosphoglycerate, which is 35% lower than that observed on HbA. The fetal variant shows an increased oxygen affinity compared with normal HbF and an almost abolished heme-heme interaction. The molecular basis of these functional differences is discussed in terms of the possible role played by the substitution of alpha (Asp-26-->His) on the stability of the R state of the molecule due to a decreased interaction at the level of alpha 1 alpha 2 contact.

[Separation of hemoglobins F, Fac, S, C, A1c and determination of hemoglobin F using high performance liquid chromatography]. / Séparation des hémoglobines F, Fac, S, C, A1c et dosage de l'hémoglobine F par chromatographie liquide haute performance.

Separation of hemoglobins F, Fac, S, C and A1c was performed using high performance liquid chromatography with a cation exchange Polycat A column in a 15-minute assay. HbF titration results were well correlated with those of the reference alkali denaturation technique for values below 12% (r = 0.95; P < 0.001). This technique may be used as a confirmation test for neonatal screening of sickle cell disease.

Rapid analysis of hemoglobin variants by cation-exchange HPLC.

We investigated the use of a 3.5 x 0.46 cm HPLC column packed with 5-microns particles of porous (100 nm) silica coated with polyaspartic acid for hemoglobin analysis. A 13-min gradient was produced between two mobile phases. The method is capable of separating more than 35 commonly encountered hemoglobin variants within 12 min. Hemoglobin variants identified include Bart's, acetyl F, H, A1c, F, Camden, N-Baltimore, J-Baltimore, N-Seattle, Grady, Fannin-Lubbock, A G-Georgia, Lepore-Baltimore, P-Galveston, G-Coushatta, Lepore-Boston, E, Osu Christiansborg, A2, G-Philadelphia, Korle Bu, Russ, Richmond, D-Los Angeles, Deer Lodge, Montgomery, S, Q-Thailand, G-San Jose, A2', Hasharon, Q-India, Tampa, GS hybrid, C-Harlem, O-Arab, British Columbia, and C. Between-run precision of an in-house pooled hemoglobin control material, AFSCA2, gave CVs of 2-5% for the A, F, S, and C and 8% for the A2 over a 6-month period. The simplicity of sample preparation, high resolution of the system, and high accuracy of the method, combined with complete automation, make this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory.

Interference with glycated hemoglobin by hemoglobin F may be greater than is generally assumed.

An automated electrophoretic method to measure glycated hemoglobin (Hb A1) was compared with manual affinity methods. Good correlation between methods was found. The electrophoretic method showed good run-to-run precision, good linearity, was free from interference by the labile aldimine fraction, and required less time and considerably less consumable expense than the affinity methods. However, as previously reported, Hb F comigrating with Hb A1 caused spurious increases in glycated Hb levels as compared with the affinity methods. This effect was linearly dependent on the Hb F concentration. Using the discrepancy between concentrations of Hb A1 by electrophoresis and glycated hemoglobin by affinity methods, 330 patients were screened in two hospitals for Hb F. A 12% frequency of elevated Hb F (defined as a level that is more than 2%) was found in patients from a community-tertiary care hospital, which is significantly greater than the 1.5% frequency commonly thought to occur in the adult population at large, whereas patients from a Veterans Administration Hospital showed an elevated frequency of 2.2%. Based on this and other studies, it is concluded that the frequency of elevated Hb F in adults may very substantially among different medical centers. The authors recommend against using this method and suggest that laboratories that persist in using it should periodically assess the frequency in patients with Hb F levels greater than 2% of total hemoglobin, replacing the method if an unacceptably high frequency is found.

Hemoglobin A1c by HPLC with the Pharmacia Mono S HR 5/N cation-exchange column: influence of sample protein load on optimal chromatographic conditions.

The Pharmacia Mono S HR 5/5 column has been optimized for hemoglobin A1c analysis by HPLC by using a much smaller column load, decreased buffer flow rate, and a steeper gradient than was used in previously described methods. Superior chromatographic separation, shorter analysis time, and a greatly extended column life have resulted.

Hb F-Cosenza or G gamma 25(B7)Gly----Glu: a new fast-moving fetal hemoglobin variant.

A fast-moving gamma chain variant was discovered in the cord blood of a newborn during a screening program carried out in the northern region of Calabria, Southern Italy. The structural analysis showed a Gly----Glu substitution at position 25 of the G gamma chain indicating a new fetal hemoglobin variant that was named Hb F-Cosenza.

Changes in alpha-globin gene expression in mice of two alpha-globin haplotypes during development.

Adult alpha-globin in mice is synthesized in large amounts during development, first in the primitive, nucleated erythrocytes of yolk sac origin and later in the definitive, nonnucleated erythrocytes that differentiate in the fetal liver, spleen, and bone marrow. Isoelectric focusing analysis of hemoglobins of mice with the Hbag2 and Hbac haplotypes shows that the ratios of alpha chain 1 to chain 5m and alpha chain 1 to chain 4 in adult hemoglobins from Hbag2 and Hbac mice, respectively, change between day 11.5 and day 16.5 of gestation in nucleated red cells, while no change occurs in nonnucleated red cells. The percentage ratios of the two different alpha-globin chains are different in Hbag2 and Hbac mice for EII, EIII, and adult hemoglobin. In nucleated red cells of yolk sac origin, differences and changes in alpha-globin ratios are a composite of changing globin gene transcription and posttranslational competitive affinities among globins to form embryonic and adult hemoglobin tetramers.