E.C. Abraham

Separation of Human Hemoglobins by Deae-Cellulose Chromatography using Glycine-Kcn-Nacl Developers

This chromatographic procedure uses DEAE-cellulose as ion exchanger and glycine-KCN-NaC1 solutions as developers. Blood samples from several adults and newborn infants with alpha, beta, delta, or gamma chains variants have been analysed. The hemoglobins are eluted as compact and symmetrical zones, and the separation of many hemoglobin types is greatly improved. The procedure is relatively fast, simple, and inexpensive.

Progressive changes in lens crystallin glycation and high-molecular-weight aggregate formation leading to cataract development in streptozotocin-diabetic rats *

Because of their remarkable longevity, lens crystallins undergo a substantial amount of glycation (non-enzymatic glycosylation) during diabetic hyperglycemia. These post-translational modifications have the potential to disrupt the structural and functional properties of the lens crystallins and contribute to the formation of cataracts. Streptozotocin-induced diabetic rats were used to study the relationship between glycation of lens proteins and the formation of insoluble high-molecular-weight (HMW) aggregates believed to be responsible for cataract formation. After the onset of diabetes, cataracts developed in about 12- to 13 weeks. The animals were followed in this manner until cataracts developed and for an additional 63 days. Five control and five diabetic rats were killed every 3 weeks and lenses removed. Levels of glycated protein and glycated amino acids in lenses from each animal were examined by affinity chromatography. In addition, the changes in crystallin composition and development of HMW aggregates were monitored by molecular-sieve HPLC techniques. As diabetic hyperglycemia continued there was a linear increase in glycated protein in both the soluble and insoluble fractions. This increase was paralleled by an increase in the soluble HMW and insoluble HMW aggregates. Other changes included a decrease in reactive sulfhydryls which indicates an increase in disulfide bond formation. The gamma-crystallin levels also decreased in a linear fashion during the hyperglycemic pre-cataract and cataract stages. It appears that the glycation of lens crystallin, the disappearance of reactive sulfhydryls and the formation of HMW aggregates are interrelated.

Differences in Affinity of Variant β Chains for a Chains: A Possible Explanation for the Variation in the Percentages of β Chain Variants in Heterozygotes

The a and β chains of the hemoglobins A, S, Leslie and N-Baltimore have been isolated as PMB derivates by CM-cellulose and DEAE-cellulose chromatography. The relative affinities of the βA, βs, βLeslie and βN-Baltimore chains for α chains were measured through quantitation by chromatography of Che hemoglobins A and Leslie, A and S, and A and N-Baltimore that were formed when variable amounts of α chains were added to a mixture of equal amounts of the appropriate β chains. The data indicate a greatly decreased affinity of βLeslie chains for a chains; a similar preference of a chains for βA chains was observed for mixtures Involving α, βA, and βs chains, but the affinity of βs chains for a chains was higher than that of βLeslie chains. The βN-Baltimore chains assembled with a chains at a similar rate as βA chains. The data as Interpreted Indicate that the affinity of certain β chains for a chains can be a major post-translatlonal control mechanism which regulates the level of a β chain variant in heterozygotes.

Advanced glycation end products in human senile and diabetic cataractous lenses

The authors prepared water-soluble (WSF), urea-soluble (USF), alkali-soluble (ASF), sonicated (SF), sonicated insoluble (SIF) and membrane (MF) fractions of lens proteins from human senile and diabetic cataractous lenses and age-matched clear lenses. Levels of advanced glycation end products (AGEs) including carboxymethyl lysine (CML), a glycoxidation product, were determined by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA). Distribution of AGEs in the various protein fractions was ascertained by SDS-PAGE and Western blotting. An overall increase in the levels of AGEs in diabetic cataractous lenses as compared to senile cataractous lenses and clear lenses has been observed. ASF and SF , both of which originated from the urea-insoluble fraction, showed the highest levels of AGEs. However, no clear-cut differences in CML levels were seen among clear lenses and senile and diabetic cataractous lenses. AGEs were found to be distributed mostly in the high molecular aggregates in all the fractions. These data suggest that AGEs contribute to protein aggregation and subsequent insolubilization.

On the chromatographic heterogeneity of human fetal hemoglobin.

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.

Identification of Hydrogen Peroxide Oxidation Sites of αA- and αB-Crystallins

The α-crystallins are the most abundant structural proteins of the lens and, because of their chaperone activity, contribute to the solubility of the other crys-tallins. With aging, the lens crystallins undergo a variety of modifications which correlate with a loss of solubility and the development of cataract. A recent study demonstrating that α-crystallins exposed in vitro to FeCl3 and H2O2 exhibit decreased chaperone activity, implicates metal catalyzed oxidations of α-crystallins in this loss of solubility. The present study has determined that α-crystallins incubated with FeCl3 and H2O2 are modified by the nearly complete oxidation of all methionine residues to methionine sulfoxide, with no other detectable reaction products. The modifications were identified from the molecular weights of peptides formed by enzymatic digestion of the α-crystallins and located by tandem mass spectrometric analysis of the fragmentation pattern of the modified peptides. A dominant pattern in the mass spectra of the frag...

New less temperature-sensitive microchromatographic method for the separation and quantitation of glycosylated hemoglobins using a non-cyanide buffer system

This paper describes a new microchromatographic method on Bio-Rex 70 ion-exchanger, which enables the isolation and quantitation of the minor components hemoglobin AIa+b and hemoglobin AIc. The method relies on the equilibration of the resin in a polyphosphate buffer with a pH closer to the pKa of the carboxylic group of the resin, and on the adjustment of the sample pH at 5. This induces linear pH and ionic strength gradient during the elution of the hemoglobin components. The method is little affected by temperature between 20 and 30 degrees C, by the presence of the aldimine Schiff base, and is not expected to be greatly influenced by moderate fluctuations in pH and ionic strength of the buffers used. There was good correlation between the values obtained by the new micromethod and four procedures currently used, namely high-performance liquid chromatography (r = 0.96), and Trivelli Bio-Rex 70 macromethod (r = 0.99), bioaffinity chromatography (r = 0.98), and Isolab hemoglobin AI kit (r = 0.91). The method is reproducible, the interassay and the intra-assay correlation coefficients did not exceed 4.2%. The mean value for hemoglobin AIc was 4.6 +/- 0.35% for eleven non-diabetics and 8.9 +/- 2.6% for twenty-one diabetics.

Differential glycation of rat α-, β- and γ-crystallins

Crystallin glycation seems to play an important role in the development of diabetic cataract. In order to understand the role of glycation in cataractogenesis, levels of glycation of different crystallins were determined by in vitro glycation of rat lens soluble fraction with 50 mm glucose or glucose-6-phosphate (G6P) for up to 5 days and in streptozotocin-diabetic rats during various stages of cataract development. All samples were reduced with [3H]NaBH4 and the tritium incorporation was taken as a measure of glycation. Proteins were routinely separated by molecular sieve HPLC. In vitro studies with glucose showed that γ-crystallin was readily glycated and reached a plateau by 3 days, while α- and β-crystallins were glycated slowly initially up to 3 days followed by a steep increase as seen on the fifth day. Incubation with 50 mm G6P resulted in an approximately two fold increase in glycation compared to glucose of all crystallins. In the diabetic animals also γ-crystallin glycation increased approximately twofold within 15 days after the onset of diabetes and an additional threefold within the next 45 days followed by a slight decrease during the following 90–120 days. Increase in glycation, on the contrary, was very slow up to 30 days for α-crystallin and up to 60 days for β-crystallin, followed by a steep increase during the remainder of the experimental period. The high molecular weight (HMW) aggregates had higher levels of glycation than other proteins; the insoluble HMW aggregates contained higher levels of glycation than the soluble HMW aggregates. Protein conformational changes may be involved in facilitating the glycation of polymeric α-and β-crystallins with increasing duration of glycation, while the monomeric γ-crystallin is readily glycated even from the beginning.

Glycation of human lens proteins: Preferential glycation of αA subunits

Abstract Glycation of crystallins and high molecular weight (HMW) aggregates was followed during aging (16–85 years) and in diabetes (44 and 70 years old). Lens soluble and insoluble fractions were reduced with [3H]NaBH4 and separated by molecular sieve HPLC. The protein content in each HPLC peak was measured by the Lowry method. The tritium incorporation, expressed as cpm mg−1 protein, was taken as a measure of early glycation and specific non-tryptophan fluorescence (Ex: 370 nm; Em: 440 nm), expressed as relative fluorescence U mg−1 protein, was taken as a measure of advanced glycation. The youngest lenses analysed were 16 and 17 years old and these provided the baseline values. The results showed that during aging there was about a three-fold increase in tritium incorporation and fluorescence of α-crystallin, while the increases in β and γ were only two-fold from the levels seen in 16- and 17-year-old lenses. On the other hand, both the soluble and insoluble HMW aggregate fractions showed up to five-fold increase in tritium incorporation during aging. The fluorescence was about two-fold higher in the insoluble HMW aggregates as compared to the soluble HMW aggregates in 16- and 17-year-old lenses and both showed an increase of about three-fold during aging. Diabetes resulted in an approximately 10–50% increase in tritium incorporation and non-tryptophan fluorescence of various crystallins and HMW aggregates. Since α-crystallins showed a higher level of glycation than other crystallins, and soluble HMW aggregates appear to have consisted predominantly of α-crystallins, we further determined the level of glycation of α-crystallin subunits. Alpha-crystallins from 50–85-year-old lenses was used to separate the subunits by both reverse-phase HPLC (RP-HPLC) and gel electrophoresis. Determination of the protein bound radioactivity showed that αA was more glycated than αB. Furthermore, the RP-HPLC also separated a modified α peak, in addition to αA and αB, which presumably originated from HMW aggregates. The modified α had an even higher level of early glycation than αA. Interestingly, the fluorescence associated with the modified α was also several fold higher than αA and αB.

An Improved Chromatographic Procedure for Quantitation of Human Fetal Hemoglobin

The DEAE-cellulose chromatographic procedure which uses glycine-KCN-NaCl solutions as Developers and a NaCl gradient to elute the hemoglobin zones was found to be useful for the quantitation of Hb F in samples containing Hb A, provided the level of Hb F exceeds 2 percent. The method has been evaluted through a study of artificial mixtures and of blood samples from patients with various disorders. The data have been compared with results obtained by four other procedures that are used for the quantitation of Hb F.