Two-dimensional analysis of glycated hemoglobin heterogeneity in pediatric type 1 diabetes patients

Interindividual and ethnic variation in glycated hemoglobin levels, unrelated to blood glucose variation, complicates the clinical use of glycated hemoglobin assays for the diagnosis and management of diabetes. Assessing the types and amounts of glycated hemoglobins present in erythrocytes could provide insight into the mechanism. Blood samples and self-monitored mean blood glucose (MBG) levels were obtained from 85 pediatric type 1 diabetes patients. Glycated hemoglobin levels were measured using three primary assays (boronate-affinity chromatography, capillary isoelectric focusing (CIEF), and standardized DCA2000+ immunoassay) and a two-dimensional (2D) analytical system consisting of boronate-affinity chromatography followed by CIEF. The 2D system separated hemoglobin into five subfractions, four of which contained glycated hemoglobins. Glycated hemoglobin measurements were compared in patients with low, moderate, or high hemoglobin glycation index (HGI), a measure of glycated hemoglobin controlled for blood glucose variation. MBG was not significantly different between HGI groups. Glycated hemoglobin levels measured by all three primary assays and in all four glycated 2D subfractions were significantly different between HGI groups and highest in high HGI patients. These results show that interindividual variation in glycated hemoglobin levels was evident in diabetes patients with similar blood glucose levels regardless of which glycated hemoglobins were measured.     

Comparison of the multiple molecular forms of bovine adrenocortical P450scc with those of corpus luteum P450scc.

1. Bovine adrenocortical P450scc was resolved into several fractions by chromatography on AH-Sepharose 4B followed by gel filtration on Toyopearl HW55S. All fractions contained P450scc of the same molecular size and the P450scc could be resolved into 3-4 major and more than 10 minor isoelectric point forms by isoelectric focusing on polyacrylamide gel in the presence of Emulgen 913. 2. Both the AH-Sepharose chromatography profile and the isoelectric focusing pattern of the adrenocortical P450scc were more complex than those of the corpus luteum P450scc. The corpus luteum P450scc was practically devoid of the neutral to acidic isoelectric point forms. 3. Three to four P450scc subfractions with different isoelectric focusing pattern were obtained from a purified preparation of adrenocortical P450scc by ion-exchange chromatography on DEAE-Toyopearl 650S or DEAE-Sephadex A25. These P450scc subfractions showed essentially the same spectral properties, catalytic activity, molecular weight and N-terminal amino acid sequence. 4. The most acidic (the latest eluting) subfraction was composed mostly of the neutral to acidic isoelectric point forms. The sedimentation characteristics of this subfraction was also studied. 5. The structural basis of the multiple molecular forms was discussed.     

A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.     

Studies on subfractions of hemoglobin A1b and hemoglobin A1c in diabetic subjects

Hemoglobin (Hb) obtained from the hemolysate of normal subjects and diabetic patients was separated into HbA1a1, HbA1a2, HbA1b, HbA1c and HbA0 (major Hb) by Bio-Rex 70 cation exchange column chromatography. The glycosylated Hbs were further separated reproductively by cation exchange high performance liquid chromatography (HPLC), using 50 mM sodium phosphate buffer pH 5.80 with 0-0.2 M NaCl linear gradient system. HbA1b and HbA1c were separated into two subfractions (HbA1b1 and HbA1b2) and three subfractions (HbA1c1, HbA1c2, HbA1c3), respectively. The percentages of each subfraction except HbA1c1 in diabetic patients were significantly higher than those in normal subjects. Furthermore, HbA1c1, HbA1c2 and HbA1c3 correlated well with fasting blood glucose levels in the prior 5 month period, while subfractions in HbA1b revealed no significant correlation with blood glucose levels. The percentages of each subfraction of HbA1c in patients either with diabetic cataracts or with diabetic neuropathy were almost the same as those in the patients without complications. However, the percentages of each of the three groups were markedly higher than those of the normal subjects. These results suggest that glycosylation of hemoglobin in diabetic patients may be increased in various sites of the molecule in parallel with the blood glucose levels during the preceding 4-5 months.     

Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites

The nonenzymatic glycosylation (glycation) of Cu-Zn-superoxide dismutase led to gradual inactivation of the enzyme (Arai, K. Iizuka, S., Tada, Y., Oikawa, K., and Taniguchi, N. (1987) Biochim. Biophys. Acta 924, 292-296). The purified superoxide dismutase from human erythrocytes comprises both glycated and nonglycated forms. The nonglycated Cu-Zn-superoxide dismutase was isolated by boronate affinity chromatography. Incubation of the nonglycated superoxide dismutase with D-[6-3H]glucose in vitro resulted in the gradual accumulation of radioactivity in the enzyme protein, and Schiff base adducts were trapped by NaBH4. The sites of glycation of the superoxide dismutase were identified by amino acid analysis after reverse-phase high performance liquid chromatography of the trypsin-treated peptides. Lysine residues, i.e. Lys3, Lys9, Lys30, Lys36, Lys122, and Lys128, were found to be glycated. Three of the glycated sites lie in Lys-Gly, two in Lys-Ala, and one in Lys-Val. The inactivation of the superoxide dismutase on the glycation is due mainly to the glycation of Lys122 and Lys128, which are supposed to be located in an active site liganding loop. The remaining five sites, such as Lys-Glu, Lys-Asp, Lys-His, and Lys-Thr are relatively inactive as to the formation of either a Schiff base or an Amadori adduct.     

Application of shielding boronate affinity chromatography in the study of the glycation pattern of haemoglobin

Human haemoglobin (Hb) may appear in a number of glycated species. The glycation pattern of Hb using shielding boronate affinity chromatography (SBAC) has been studied in the present work. SBAC is a novel separation technique, which eliminates nonspecific boronate-protein interactions by introducing a so-called shielding reagent. Two samples from Bio-Rad (Lyphochek)--one from normal persons' blood with relatively low HbA(1c) level (HbL) and the other from diabetic patients' blood with an elevated HbA(1c) level (HbH)--were used for the investigation. Glycated Hb (GHb) was separated from nonglycated Hb species using Tris as the shielding reagent. Two eluted peaks, eluted peak 1 (E1) and eluted peak 2 (E2), were obtained using a linear gradient elution with Tris. Several bands were observed on isoelectric focusing gel, which showed the same migration positions as Hb adducts, such as HbA(0), which is major Hb component containing two alpha chains and two beta chains; HbA(1c), which is post-translational glycation on the N-terminus of the beta chains of HbA(0); Foetal Hb (HbF), consisting of two alpha chains and two gamma chains; and glutathione Hb (also called HbSSG), which is the result from thiol-disulphide interchain exchange during oxidation of the thiol groups of Hb. In both HbL and HbH samples, E2 exhibited slightly higher amounts of HbF than E1. Electrospray-ionisation mass spectrometry showed that: (1) HbL-E1 was glycated with single glucose on both alpha and beta chains while no observable glycated chains were present in HbL-E2; (2) both HbH-E1 and HbH-E2 were glycated with single glucoses on both alpha and beta chains, however, compared with HbH-E1, HbH-E2 showed a higher relative intensity of the glycated beta chain and lower relative intensity of the glycated alpha chain; and (3) the degree of glycation increased with increasing glycation level of the sample. The amount of HbA(1c) presented in the eluted peaks was further determined using enzymatic digestion of glycated Hb by endoproteinase Glu-C and the subsequent separation and analysis of the digested peptides by reversed-phase high-performance liquid chromatography and capillary electrophoresis. The values of HbA(1c)/HbA(0) of the eluted peaks, i.e. HbL-E1, HbL-E2, HbH-E1 and HbH-E2, were 0.27, 0.19, 0.50 and 0.43, respectively. In both HbL and HbH samples, E1 contained higher amounts of HbA(1c) than E2. This study demonstrates the structural heterogeneity of GHb as well as the possibility of using SBAC to detect glycated species of Hb.     

Study of the relationship between the subfraction composition of histone F1 and the type of tissue in birds.

The subfraction composition of lysine-rich histone has been studied with the aid of polyacrylamide gel electrophoresis. The subfraction compositions of the histone F1 of several tissues from the chicken, pigeon, and titmouse have been compared. The histone F1 from the tissues investigated consists of four or five subfractions of similar number and electrophoretic mobility (1, 1a, 2, 3, and 4). In the different avian species each subfraction varied its mobility independently of the others. The chicken tissues investigated can be divided into two classes, depending on the relative concentration of subfractions 2 and 3 (A and B): Class A (subfraction 2 is smaller than 3) includes the brain, liver, skeletal muscle, heart, muscular layer of the stomach, and pancreas, and class B (subfraction 2 is larger than 3) includes the intestinal mucosa, thymus, and testes, as well as the liver, heart, and pancreas from a 21-day embryo. Such a division of the tissues corresponds to the varying rate of their cellular renewal. In a parallel examination of the relative concentrations of the individual subfractions in the same tissues from the three avian species it has been found that the relative concentration of subfractions 3 and 2 is increased in the skeletal muscles, heart, brain, and liver, that subfraction 2 is increased in the intestinal mucosa, that subfractions 4 and 3 are increased in the pancreas, and that subfractions 1, 1a, and 4 are increased in the erythrocytes. The results obtained may be interpreted as a consequence of some relationship between the subfraction composition of histone F1 and the type of tissue of the source.     

Effect of phosphate on the kinetics and specificity of glycation of protein

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffers in order to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues in or near the active site. In contrast, in the cationic buffers, 3-(N-morpholino)propane-sulfonic acid and 3-(N-tris(hydroxymethyl)methyl-amino)-2-hydroxypropanesulfonic acid, the kinetics of glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNAse was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increased glycation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effects on the glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establish that buffering ions or ligands can exert significant effects on the kinetics and specificity of glycation of proteins.     

Chromato-focussing of plasma low density proteins

A few alternatives of the binding of healthy patients plasma low density lipoproteins (LDL) with anion exchanger PBE-94 were revealed. In the first case the main part of LDL did not bind to the gel and the isoelectric points of the minor subfractions were 5.8 and 5.3, and pI 4.1. In the second case about half of lipoproteins did not bind to the gel, and the isoelectric points of subfractions were 5.7 and 5.0; and pI 4.1. In the third case when all lipoproteins bound to the PBE-94, there were much more subfractions and their isoelectric points were 6.2, 5.8, 5.2, 4.9, 4.5 and pI 4.1. All LDL of the patients with ischemic heart disease bound to anion exchanger, and the part of subfraction with pI 4.1 was three or five times as great as the one of the healthy person. Increasing of the LDL subfraction with pI 4.1 was observed at prolonged keeping of the LDL obtained from the healthy person plasma. LDL isoelectric point distribution of the persons with carcinoma uterine cervix did not differ from the one of the healthy persons. Acetylation and hexanol modification resulted in the isoelectric point shift from 5.7 to 4.6 and to 4.3 in the case of LDL subfraction to be obtained preparatively using the chromatofocusing.     

ATP-dependent Mechanism Protects Spectrin against Glycation in Human Erythrocytes

Human erythrocytes are continuously exposed to glucose, which reacts with the amino terminus of the β-chain of hemoglobin (Hb) to form glycated Hb, HbA1c, levels of which increase with the age of the circulating cell. In contrast to extensive insights into glycation of hemoglobin, little is known about glycation of erythrocyte membrane proteins. In the present study, we explored the conditions under which glucose and ribose can glycate spectrin, both on the intact membrane and in solution and the functional consequences of spectrin glycation. Although purified spectrin could be readily glycated, membrane-associated spectrin could be glycated only after ATP depletion and consequent translocation of phosphatidylserine (PS) from the inner to the outer lipid monolayer. Glycation of membrane-associated spectrin led to a marked decrease in membrane deformability. We further observed that only PS-binding spectrin repeats are glycated. We infer that the absence of glycation in situ is the consequence of the interaction of the target lysine and arginine residues with PS and thus is inaccessible for glycation. The reduced membrane deformability after glycation in the absence of ATP is likely the result of the inability of the glycated spectrin repeats to undergo the obligatory unfolding as a consequence of interhelix cross-links. We thus postulate that through the use of an ATP-driven phospholipid translocase (flippase), erythrocytes have evolved a protective mechanism against spectrin glycation and thus maintain their optimal membrane function during their long circulatory life span.     

Electrochemical detection of HbA1c, a marker [correction of maker] for diabetes, using a flow immunoassay system

An on-chip electrochemical flow immunoassay system for the detection of hemoglobin A1c (HbA1c) was developed using anti-human hemoglobin (Hb) IgG labeled with ferrocene monocarboxylic acid (Fc-COOH) and boronate-affinity chromatography. An on-chip column packed with boronate-activated agarose beads was used for the separation of HbA1c from both non-glycated Hb and free antibody. Anti-human Hb IgG conjugated to Fc-COOH (Fc-IgG) was used for the electrochemical detection of HbA1c. The assay procedure included immunoreactions with Fc-IgG and HbA1c, separation of immunocomplexes by boronate affinity, and electrochemical detection of Fc-IgG-HbA1c immunocomplexes. The immunoreaction mixtures were injected onto a boronate-affinity column. HbA1c-antibody complexes were then trapped onto the column by the affinity of HbA1c to boronic acid. Subsequently, elution buffer containing sorbitol was applied to elute HbA1c-antibody complexes and a current was detected by applying 600 mV versus Ag/AgCl. The elution signal was an estimation of the HbA1c amount. A linear correlation between the increase of current and HbA1c concentration was obtained up to an HbA1c concentration of 500 microg/ml. The HbA1c flow immunoassay was successfully achieved using hemolysates. This electrochemical flow immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of glycated proteins including HbA1c.     

Markers of glycemic control in the mouse: comparisons of 6-h- and overnight-fasted blood glucoses to Hb A1c

The present studies examined the relationship between fasting blood glucose and Hb A(1c) in C57BL/6J, DBA/2J, and KK/HlJ mice with and without diabetes mellitus. Daily averaged blood glucose levels based on continuous glucose monitoring and effects of 6-h vs. overnight fasting on blood glucose were determined. Daily averaged blood glucose levels were highly correlated with Hb A(1c), as determined with a hand-held automated device using an immunodetection method. R(2) values were 0.90, 0.95, and 0.99 in KK/HIJ, C57BL/6J, and DBA/2J, respectively. Six-hour fasting blood glucose correlated more closely with the level of daily averaged blood glucose and with Hb A(1c) than did blood glucose following an overnight fast. To validate the immunoassay-determined Hb A(1c), we also measured total glycosylated hemoglobin using boronate HPLC. Hb A(1c) values correlated well with total glycosylated hemoglobin in all three strains but were relatively lower than total glycosylated hemoglobin in diabetic DBA/2J mice. These results show that 6-h fasting glucose provides a superior index of glycemic control and correlates more closely with Hb A(1c) than overnight-fasted blood glucose in these strains of mice.     

Changes in subfraction composition of chicken lysine-rich histone during ontogenesis]

Lysine-rich histone isolated from different chicken tissues was separated electrophoretically into 4-5 subfractions. The subfrations reffered to as 1, 2, 3, and 4, occur in each the tissue studied, erythrocyte lysine-rich histone containing an additional subfraction 1a. F1 histone from mitotically active tissues (intestinal mucosa, thymus, testes) has a higher content of subfraction 2, while the same histones from mototically inactive tissues (liver, heart, brain) contain an elevated amount of subfraction 3. F1 histone isolated from liver, brain and heart of 21-day embryo has much more of subfraction 2, than the same histone of adult animal. During the chicken development from hatching till maturation the content of subfraction 2 in these organs decreases, and the content of subfraction 3 increases. The rate of this change in liver corresponds to the rate of DNA synthesis. In F1 histone of erythrocytes the content of subfraction 4 falls down during the post hatching ontogenesis.     

Isolation and functional characterization of hemoglobin Casper: beta106(G8) Leu replaced by Pro.

Hemoglobin Casper (beta106Leu replaced by Pro) can be separated from hemoglobin (Hb) A by isoelectric focusing on polyacrylamide gel. This abnormal hemoglobin was estimated to be 30% of teh total by both isoelectric focusing and heat lability kinetics. Its oxygen equilibrium curves indicate a high oxygen affinity, low degree of subunit interaction, and a decreased Bohr effect. Mixtures of Hb Casper and Hb A appear to bind oxygen as if no hybrid molecules exist.     

Further resolution of adult chick hemoglobins by isoelectric focusing in polyacrylamide gel.

Evidence is presented that adult chick hemoglobins exist in four types separable by isoelectric focusing on polyacrylamide gels instead of the two hemoglobin types previously resolved by other methods. These are hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin D1 (HbD1), and hemoglobin D2(HbD2). Their pI values are 7.53 +/- 0.02, 7.37 +/- 0.02, 6.92 +/- 0.04 and 6.72 +/- 0.05, respectively, constituting about 63, 14, 18 and 5% of the total hemoglobin from adult chick erythrocytes, respectively. HbA1 and HbA2 ar identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weight and amino acid composition of HbD1 and HbD2 are also identical although there are differences in their tryptic peptides. Experiments were done to show that the existence of four hemoglobin types is not due to genetic heterogeneity of the experimental animal, nor to artifacts of oxidation of carboxyhemoglobin to methemoglobin tetramers. Care was exercised to eliminate deamination and modification of side chain amino groups by using freshly prepared hemolysates and to minimize the "plateau phenomenon" peculiar to isoelectric focusing by controlling the duration of electrophoresis. The use of cyanmet form of (thus liganded) hemoglobin in this study reduced the chance of heterotetramer formation. Furthermore, consideration was given to possible anomalies caused by ampholytes. In the face of negative evidence for artifacts, it is concluded that adult chicken has more than the two hemoglobin types previously reported.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.     

Acute Coronary Syndrome Remodels the Protein Cargo and Functions of High-Density Lipoprotein Subfractions

Objectives

This study examined alterations in the functions and proteome of high-density lipoprotein (HDL) subfractions (HDL2 and HDL3) isolated from patients with acute coronary syndrome (ACS) compared with control subjects.

Methods

We measured HDL subfraction cholesterol efflux capacity, inflammatory index (HII), paraoxonase-1 (PON1) activity, and lipid hydroperoxide (LOOH) levels in both male age-matched controls and the ACS group (n = 40/group). Additionally, proteomic analysis was used to monitor changes in the HDL subfraction proteome between controls and ACS subjects.

Results

Both HDL2 and HDL3 from ACS patients had greater HII and LOOH levels compared with controls (P<0.001); PON1 activity and cholesterol efflux capacity in both HDL2 and HDL3 from the ACS group were significantly less than those of controls (P<0.001). Using proteomic analysis, we demonstrated that, compared with the control group, nine proteins were selectively enriched in HDL3 from subjects with ACS, and ras-related protein Rab-7b was decreased in HDL3. Additionally, in the ACS subjects, 12 proteins were decreased in HDL2 and 4 proteins were increased in HDL2.

Conclusions

Functional HDL subfractions shifted to dysfunctional HDL subfractions during ACS, and the functional impairment was linked to remodeled protein cargo in HDL subfractions from ACS patients.     

Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection

A modified version of capillary isoelectric focusing (cIEF) was developed to separate hemoglobin variants contained within single human erythrocytes. Laser-induced native fluorescence with 275 nm excitation was used to detect the separated hemoglobins. In this method, baseline fluctuations were minimized and detection sensitivity was improved by using dilute solutions of anolyte, catholyte, and carrier ampholytes (with methylcellulose). Since electrosmotic flow was used for mobilization of the focused bands, separation and detection were integrated into a single step. The capillary was first filled with only ampholyte solution, and the cell (or standard) was injected as in capillary zone electrophoresis. The ∼90 fl injection volume for individual cells is 7×10⁴ times lower than those previously reported. Adult (normal and elevated A₁), sickle (heterozygous), and fetal erythrocytes were analyzed, with the amounts of hemoglobins A₀, A_1c, S and F determined. The pH gradient for cIEF was linear (r² = 0.9984), which allowed tentative identification of Hb F_ac. Variants differing by as little as 0.025 pI units were resolved.     

Characteristics of membrane protein phosphorylation in Plasmodium berghei-infected mouse erythrocytes

Membrane protein phosphorylation in Plasmodium berghei-infected erythrocytes was studied by incubating intact cells with (32P)orthophosphate and incubating isolated membrane with (gamma-32P)ATP. Phosphorylated proteins were detected by autoradiography after sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis or isoelectric focusing followed by gel electrophoresis. New phosphorylated proteins were found in membrane from infected erythrocytes, including a protein with electrophoretic mobility identical to band 5, with Mr 43,000. The molar ratio of phosphate to protein ranged between 0.1 and 0.5. Isoelectric focusing-SDS polyacrylamide gel electrophoresis, peptide mapping, extractability properties, and reduction of susceptibility to DNase I inhibition suggested that this protein is phosphorylated actin. In contrast, spectrin phosphorylation in infected erythrocytes was mostly unchanged.