Rapid preparation of native alpha and beta chains of human hemoglobin.

1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is less than 0.1%. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.     

Bioflavonoids as poisons of human topoisomerase II alpha and II beta

Bioflavonoids are human dietary components that have been linked to the prevention of cancer in adults and the generation of specific types of leukemia in infants. While these compounds have a broad range of cellular activities, many of their genotoxic effects have been attributed to their actions as topoisomerase II poisons. However, the activities of bioflavonoids against the individual isoforms of human topoisomerase II have not been analyzed. Therefore, we characterized the activity and mechanism of action of three major classes of bioflavonoids, flavones, flavonols, and isoflavones, against human topoisomerase IIalpha and IIbeta. Genistein was the most active bioflavonoid tested and stimulated enzyme-mediated DNA cleavage approximately 10-fold. Generally, compounds were more active against topoisomerase IIbeta. DNA cleavage with both enzyme isoforms required a 5-OH and a 4'-OH and was enhanced by the presence of additional hydroxyl groups on the pendant ring. Competition DNA cleavage and topoisomerase II binding studies indicate that the 5-OH group plays an important role in mediating genistein binding, while the 4'-OH moiety contributes primarily to bioflavonoid function. Bioflavonoids do not require redox cycling for activity and function primarily by inhibiting enzyme-mediated DNA ligation. Mutagenesis studies suggest that the TOPRIM region of topoisomerase II plays a role in genistein binding. Finally, flavones, flavonols, and isoflavones with activity against purified topoisomerase IIalpha and IIbeta enhanced DNA cleavage by both isoforms in human CEM leukemia cells. These data support the hypothesis that bioflavonoids function as topoisomerase II poisons in humans and provide a framework for further analysis of these important dietary components.     

The alpha chains of goat hemoglobins: old and new variants in native Apulian breeds

Blood samples were collected from 324 goats belonging to the native Apulian breeds Garganica and Jonica; 60 Alpine goats were also sampled to serve as a comparison. Hemoglobin phenotypes were analyzed with isoelectric focusing in a pH range of 6.7-7.7. Heterogeneity of globin chains was evidenced both by AUT-PAGE and RP-HPLC. The primary structure of four alpha globins was analyzed by combined mass spectrometry approaches. Two of these globins had never been sequenced before. One was a new alpha variant, an allele of the HBA1A gene from which it differed for the mutation A26T and has been registered with a low frequency only in Apulian breeds; the other was a globin encoded by the HBA2 locus, whose primary structure was previously derived from the corresponding gene. The two alleles recorded at the HBA2 locus presented a different frequency in the three breeds but may be considered to be generally rather common. Notwithstanding the sample size no goat was found to exhibit HbA1B. The authors discuss their findings in the light of the results reported by other researchers and argue that, in spite of what had been inferred in pioneer works on goat hemoglobins, HBA1B is not a common allele.     

Sequence determinants of nuclear localization in the alpha and beta isoforms of human topoisomerase II.

The alpha and beta isoforms of DNA topoisomerase II (topo II) are targets for several widely used chemotherapeutic agents, and resistance to some of these drugs may be associated with reduced nuclear localization of the alpha isoform. Human topo IIalpha contains a strong bipartite nuclear localization signal (NLS) sequence between amino acids 1454 and 1497 (alphaNLS(1454-1497)). In the present study, we show that human topo IIalpha tagged with green fluorescence protein is still detectable in the nucleus when alphaNLS(1454-1497) has been disrupted. Seven additional regions in topo IIalpha containing overlapping potential bipartite NLSs were evaluated for their nuclear targeting abilities using a beta-galactosidase reporter system. A moderately functional NLS was identified between amino acids 1259 and 1296. When human topo IIbeta was examined in a similar fashion, it was found to contain two strongly functional sequences betaNLS(1522-1548) and betaNLS(1538-1573) in the region of topo IIbeta comparable to the region in topo IIalpha that contains the strongly functional alphaNLS(1454-1497). The third, betaNLS(1294-1332), although weaker than the other two beta sequences, is significantly stronger than the analogous alphaNLS(1259-1296). Differences in the NLS sequences of human topo II isoforms may contribute to their differences in subnuclear localization.     

Distinct genotype and antigenicity among genogroup II sapoviruses

SaV, a pathogen of acute gastroenteritis, is divided into five genogroups, GI to GV. However, the relation between SaV antigenicity and genetic clusters is not fully understood. We have recently identified two GII SaV strains, Mc10 and C12, which are grouped into the same cluster based on the polymerase but are grouped into distinct clusters based on the capsid. To evaluate the difference in antigenicity between these two strains, VLP were expressed in mammalian cells. An antigen ELISA demonstrated for the first time that strains in the same GII SaV genogroup, but within different clusters, have distinct antigenicities.     

Amino-acid sequences of the alpha and beta chains of adult hemoglobins of the Grand Galago, Galago crassicaudatus

The adult Grand Galago (Galago crassicaudatus) was found to have two hemoglobin components (Hb I and Hb II) which were separated by carboxymethyl cellulose column chromatography. The alpha and beta chains of each component were isolated. The tryptic peptides of the alpha and beta chains were each isolated and sequenced by the conventional method. The alignment of these peptides in each chain was deduced from the homology of their sequences with that of human adult hemoglobin. The alpha chains from Hb I and Hb II were considered to be identical. On the other hand, there was only one amino-acid difference between the two beta chains at the 125th residue from the N-terminus.     

Oxygen equilibria of hybrid-heme hemoglobins containing proto- and mesoheme groups. On the nonequivalence of alpha and beta chains.

Hybrid-heme hemoglobins, alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2, were prepared, and the O2 equilibria of their alpha and beta chains were measured separately at the isosbestic points of the partner chains at different pH values and in the presence and absence of inositol hexaphosphate. The Adair equation was extended to distinguish between the O2 saturations of the alpha and beta chains, and the seven equilibrium parameters were obtained by curve fitting to those equations. The results showed that the beta chains have an affinity slightly higher than the alpha chains in the binding of the first O2 molecule. For the second O2 molecule, the molecular species that has been oxygenated on the alpha chain has a higher affinity than that carrying O2 on the beta chain. The slopes of the Hill plots were higher for the alpha chain. The O2 saturation curves for the alpha and beta chains were calculated from the parameters averaged for the hybrids alpha(meso)2beta(proto)2 and alpha(proto)2beta(meso)2 in order to cancel the effects of the heme replacement. The curves showed that the difference in O2 saturation between the two kinds of chains depends on the conditions and on the degree of O2 saturation. It was concluded that the functional difference between the chains is small enough so that it is not required to modify the models already accepted for the cooperativity of hemoglobin.     

Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.     

Bovine follitropin. Isolation and characterization of the native hormone and its alpha and beta subunits

1) A reproducible procedure was developed for the purification of bovine follitropin. 2) The method involved ammonium sulfate precipitation, ion exchange and adsorption chromatography, concanacaline-A-Sepharose chromatography and gel filtration. 3) A specific radioligand receptor assay was used to monitor each chromatographical step. 4) The potency of highly purified bovine follitropin as measured by Steelman and Pohley bioassay was 62 times the NIH-FSH-B1 standard preparation. 5) Contaminations of bovine follitropin by other glycoprotein hormones such as thyrotropin and lutropin amounted to 3 and 0.45 per cent by weight respectively as measured by specific radioimmunoassays and radioligand receptor assays. 6) The subunits alpha and beta of bovine follitropin were obtained by incubation in acidic urea, the chains being then separated by anion exchange chromatography. The subunits were subjitted to complete characterization. The amino-terminal residue of the alpha subunit is phenylalanine while a half cystine residue was found at the aminoterminal end of the beta chain. 8) Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassays and amounted to 0.02 and 0.1 per cent by weight respectively.     

Hemoglobins Austin and Waco: two hemoglobins with substitutions in the alpha 1 beta 2 contact region.

Hemoglobins (Hbs) Austin and Waco were detected by their electrophoretic migration on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). By these methods, both variants migrated between Hbs A and F. Globin chain analysis at pH 8.6 indicated that the mutant β chain of Hb Austin was faster moving than the β^A chain; however, the mutant chain of Hb Waco was indistinguishable from the β^A chain by this technique. The two variants were isolated by ion-exchange column chromatography. Sequence studies demonstrated a substitution of serine (Hb Austin) and lysine (Hb Waco) for arginine at position 40 in the β chain. These mutations involve an amino acid residue in the α₁β₂ contact region, which, before this report, had been considered invariant in all hemoglobin sequences. Hb Austin was found to exist as dimers when oxygenated and as tetramers when deoxygenated. The equilibrium constant (K_d) for the tetramer to dimer transition was approximately 300 × 10^?6m, as calculated from sedimentation velocity studies. This variant also had high oxygen affinity, a much reduced heme-heme interaction, and a normal Bohr effect. The functional properties of Hb Waco were similar to those of Hb A.     

The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)