Hemoglobin Peptide Research Articles

Induction of Trypanosoma cruzi Metacyclogenesis in the Gut of the Hematophagous Insect Vector, Rhodnius prolixus, by Hemoglobin and Peptides Carrying αD-Globin Sequences

Trypanosoma cruzi, a protozoan responsible for the American trypanosomiasis (Chagas disease), multiplies and differentiates in the gut of triatomine insect vectors. The effects of hemoglobin and synthetic peptides carrying αD-globin fragments on both the growth and the transformation of T. cruzi epimastigotes (noninfective) into metacyclic trypomastigotes (infective forms) were studied. This differentiation in the insect′s gut is expressed when hemoglobin and synthetic peptides corresponding to residues 30-49 and 35-73 of the αD-globin were added to the plasma diet. However, synthetic peptide 41-73 does not induce differentiation of epimastigotes even in the presence of the two former synthetic peptides. Thus, these data delineate an unusual molecular mechanism which modulates the dynamics of transformation of epimastigotes into metacyclic trypomastigotes in the triatomine vector′s gut.

Two missense mutations in the beta-globin gene can cause severe beta thalassemia. Hemoglobin Medicine Lake (beta 32[B14]leucine-->glutamine; 98 [FG5] valine-->methionine).

We studied the molecular basis of transfusion-dependent hemolytic anemia in an infant who rapidly developed the phenotype of beta thalassemia major. DNA sequence of one beta-globin gene of the proband revealed two mutations, one for the moderately unstable hemoglobin (Hb) Köln and another for a novel codon 32 cytosine-thymidine-guanine-->cytosine-adenine-guanine transversion encoding a leucine-->glutamine mutation. A hydrophilic glutamine residue at beta 32 has an uncharged polar side chain that could potentially distort the B helix and provoke further molecular instability. This new hemoglobin was called Hb Medicine Lake. Biosynthesis studies showed a deficit of beta-globin synthesis with early loss of beta-globin chains. An abnormal unstable hemoglobin, globin chain, or tryptic globin peptide was not present, demonstrating the extreme lability of this novel globin. Hb Medicine Lake mRNA was present, but an aberrantly spliced message was not. Absence of an abnormal beta-globin gene in the mother makes it likely that a de novo mutation occurred in the proband. The molecular pathogenesis of Hb Medicine Lake illustrates a mechanism whereby the phenotype of a genetic disorder, like the mild hemolytic anemia associated with a hemoglobinopathy, can be modulated by a coincident mutation in the same gene.

Diode laser-based concentration gradient imaging detector for capillary isoelectric focusing

A diode laser-based, real-time, universal concentration gradient imaging detector was constructed and tested for capillary isoelectric focusing (CIEF). The detector indicates sample zones by measuring refractive index gradients created by the concentration gradients of samples. An imaging detection system is the best detection approach for CIEF because all samples are focused inside the capillary column after separation. A diode laser was found to be an ideal light source for the imaging detector because of its small size, low cost, and light beam characteristics. The detector can be operated at any wavelength and does not need a high-intensity light beam. In order to focus the non-circular, non-Gaussian diode laser beam through the capillary, the optical geometry of the imaging detector was optimized. Because of the short analysis time (2–4 min), good reproducibility for determining isoelectric points, and the universality of the detector, the CIEF-imaging detector system has many possible applications, such as analysis of human hemoglobin variants and peptide mapping. This is the first report of a diode laser-based imaging detection system for analytical chemistry.

Antagonism of superantigen-stimulated helper T-cell clones and hybridomas by altered peptide ligand.

T-cell activation by an immunogenic peptide can be antagonized by nonstimulatory analogs of that peptide. We investigated this T-cell receptor antagonism by using staphylococcal enterotoxin superantigen to stimulate hemoglobin-specific helper T (Th) cells because its activation pathway may differ from that of conventional antigen. Interestingly, superantigen activation of these Th cells was antagonized by hemoglobin peptide analogs even though agonist (superantigen) and antagonist (analog peptide) bind at different sites on the major histocompatibility complex-encoded molecule and the T-cell receptor. The antagonism appeared to be a fundamental block in T-cell activation, as phosphoinositol generation, cytokine production, and proliferation were reduced in Th1 clones, and, similarly, proliferative and cytokine responses were inhibited in Th2 cells. Even T-cell hybridoma activation (cytokine production and apoptosis) was inhibited by peptide antagonists. Furthermore, analog peptides that functioned as partial agonists for these Th cells also antagonized superantigen-induced proliferation and thus were a subset of the peptide antagonists. In summary, our results demonstrate that analogs of immunogenic peptide are potent antagonists for Th cell responses induced by superantigen as well as immunogenic peptide.

Characteristics of peptides which compete for presented antigen‐binding sites on antigen‐presenting cells

The T cell recognition of globular protein antigens requires the cell surface presentation of the protein by Ia-expressing antigen-presenting cells (APC). The mechanisms by which APC function remain to be elucidated. To gain a better understanding of association of antigen with APC surfaces, a large panel of peptides of diverse physicochemical properties was assayed for the ability to compete with presented antigen for binding sites on the APC surface. Competition was measured by the ability of a peptide to block the I-Ek-restricted T cell response to pigeon cytochrome c (Pc) as presented by APC. The panel assayed included overlapping peptides representing the entire length of sperm whale myoglobin and the alpha and beta chains of human adult hemoglobin as well as synthetic conformational peptides of lactate dehydrogenase C4 exhibiting stable secondary, alpha-helical structures. The results presented here show that several peptides of this group compete with the presented form of Pc for binding sites on the APC. However, there is no single biochemical property or amino acid sequence algorithm which predicts the blocking ability. The peptides which compete with presented Pc are not predicted to assume the amphipathic alpha-helical conformation hypothesized by De Lisi and Berzofsky (Proc. Natl. Acad. Sci. USA 1986. 82: 7048) for T cell antigenic peptides. However, peptides designed and synthesized to adopt a stable alpha-helical secondary structure show more potent blocking activity than the corresponding linear peptides, suggesting that the secondary structure may indeed be a contributing factor in the ability of presented antigenic peptides to be bound by the APC. The results with the myoglobin and hemoglobin peptides show no connection between any particular secondary structure of the peptide in the native proteins and the ability of the peptides to block presentation. Further, there is no correlation between the major histocompatibility complex restriction of the competing peptides and their ability to block the I-Ek-restricted Pc-specific T cell response. This suggests that antigen presented by the APC may be bound to APC structures other than Ia prior to association with Ia. Such additional binding sites for presented antigen may be necessary to facilitate association with Ia.

Structure, Antigenic Determinants of Some Clinically Important Insect Allergens: Chironomid Hemoglobins

Determination of the molecular structure and properties of allergens that elicit severe immediate-type hypersensitivity diseases in humans and a knowledge of the structure of their antibody-binding sites should provide new insight into the pathogenetic mechanisms of allergic diseases. Monomeric and homodimeric hemoglobins (CTT I to X) have been identified as potent allergenic components of Chironomidae, a family of Diptera. Immunologic investigations of peptides of three of these hemoglobins (CTT IV, CTT VI, and CTT VIII) showed that human antibodies of the E and G classes recognize at least two different sites within each molecule. Individual hemoglobin peptides were aligned with homologous regions of chironomid hemoglobin CTT III, whose tertiary structure has been determined by x-ray analysis at a resolution of 1.4 angstroms. The antigenic site CTT IV(91 to 101) showed the following characteristics: (i) seven polar or hydroxylated amino acids, from a total of eleven, occupying predominantly superficial regions; (ii) the property of linkage to other molecules by hydrogen bonds or solvent clusters; and (iii) high thermal mobility factors. In contrast, peptide CTT IV(102 to 108), which does not bind human antibodies, contained no polar amino acids and had low thermal mobility factors. These results support the idea that the antigenicity of clinically relevant proteins is related to regions with a predominance of polar amino acids and with low energy barriers between different conformations, which allow high flexibility, including site-specific adaptation in antibody binding.

Degradation of peptides and proteins of different sizes by homogenates of human MRC5 lung fibroblasts: Aged cells have a decreased ability to degrade shortened proteins

The degradation of haemoglobin and haemoglobin-derived peptide fragments by homogenates of MRC5 fibroblasts has been investigated. Results show that the smaller fragments were degraded more rapidly than larger substrates at both pH 5.5 and pH 7.5. Only the smallest of the soluble cyanogen bromide peptides ( M r 3500) was degraded at pH 7.5. Degradation at pH 5.5 proceeded more rapidly than that at pH 7.5 for all substrates tested but was more marked with the larger substrates. Homogenates prepared from aged cells degraded puromycyl peptides and, to a lesser extent, cyanogen bromide peptides at a slower rate, at pH 7.5, than those prepared from younger cells. We suggest that cytosolic degradation is less selective and at least one cytosolic proteolytic activity decreases as cells age.

A Comparative Study of the Separation of the Tryptic Peptides of the β-Chain of Normal and Abnormal Hemoglobins by Reversed Phase High Performance Liquid Chromatography

Abstract The separation of the tryptic peptides of the human hemoglobin A β-chain by reversed phase high performance liquid chromatography under different elution conditions on several microparticulate alkylsilica supports is described. Similar methods have been used to separate the tryptic peptides of β-chain hemoglobin variants including HbC, HbE, and Hb (Kempsey). Selectivity differences which can be achieved under the different chromatographic conditions have been exploited to permit the assignment of all the anticipated peptide fragments derived from the tryptic digestion of these β-chain Hb-variants.

Renin-like and cathepsin D activities in bovine pineal glands.

Pineal extract is shown to contain both renin-like and cathepsin D activities. Evidence of renin-like activity in the bovine pineal gland was brought by incubation with natural and synthetic renin substrates and by inhibition by pepstatin. Cathepsin D activity was demonstrated by incubation with hemoglobin and synthetic fluorogenic peptide. The separation of both activities was performed by affinity chromatography on a caseinyl-Sepharose gel. The elution of the extract on affinity chromatography allowed to separate the renin-like activity, which is not retained by the gel at acid pH, from cathepsin D activity, which binds to the column at acid pH and is eluted at alkaline pH. The isolated pineal renin-like activity was found higher on tetradecapeptide renin substrate than on angiotensinogen at pH 5.5. The pH dependence of pineal renin-like activity showed two peaks of activity. One broad peak between pH 6 and 8 and one sharp peak at pH 3.5-4. These results demonstrate the existence of renin-like and cathepsin D activities in bovine pineal gland. They suggest moreover that the renin-like activity measured might represent a mixture of at least two different enzymatic activities.

Nonenzymatic glycosylation of peripheral nerve protein in diabetes mellitus.

A new affinity chromatography system that selectively retains glycosylated amino acids has been utilized to determine the amount of nonenzymatic glycosylation present in peripheral nerve from diabetic and control rats and dogs. The mean value for glycosylated amino acids in diabetic rats was 2.8 times greater than the mean value in normal rats (P less than 0.001). In diabetic dogs, mean values were 2.15 times greater than normal values (P less than 0.05). Amino acid analysis of reduced, glycosylated amino acids previously isolated by affinity chromatography showed that glycosylated lysine and its hydrolysis rearrangement products were the major borohydride-reducible adduct present. In addition, another glycosylated product was noted to be present in major proportions. This radioactive product did not chromatograph with any of the available glycosylated amino acid standards. The finding that diabetes results in a nearly 3-fold increase of peripheral nerve glycosylation is consistent with a number of previous investigations in which glycosylation was measured in hemoglobin, serum albumin, and urinary amino acids and peptides from diabetics and normals. The results reported here provide evidence that increased nonenzymatic glycosylation is occurring in a tissue where physiological, morphological, and clinical degeneration characteristically develop as a result of diabetes mellitus.

Field desorption mass spectra of tryptic peptides of human hemoglobin chains

Field desorption mass spectra of tryptic peptides of normal human hemoglobin α-, β-, γ-, δ-chains and abnormal β-chain of hemoglobin S were studied. Almost all mass peaks of the protonated molecular ions of tryptic peptides and many doubly charged ions were observed. The molecular weights of all tryptic peptides were estimated from the mass spectra, the heaviest mass being m/z 2955, and coincided with the values obtained from the known amino acid sequences. The different chains of hemoglobin can be distinguished by their field desorption mass spectra. The mass spectrum of abnormal β-chain of hemoglobin S (sickle-cell-anemia) showed a shift of the characteristic line due to the mutation of amino acid.

Separation of hemoglobin peptides by high performance liquid chromatography (HPLC).

(1980). Separation of Hemoglobin Peptides by high Performance Liquid Chromatography (HPLC) Hemoglobin: Vol. 4, No. 3-4, pp. 551-559.

Separation of the Aγ and Gγ cyanogen bromide peptides of human fetal hemoglobin by high-pressure liquid chromatography

The G γ and A γ variants of human fetal hemoglobin are defined on the basis of a single glycine/alanine substitution at position 136 of the γ chain which is located in the carboxyterminal cyanogen bromide fragment (γCB-3). This communication describes the rapid separation of these two tridecapeptides by high-pressure liquid chromatography. Application of this new technique will facilitate the study of the biosynthesis and genetics of human fetal hemoglobin.

A comparison of the tryptic peptides of hemoglobin from two cricetine genera: Peromyscus and Calomys

The composition of tryptic peptides was determined for the major hemoglobin from two cricetine rodents. Peromyscus maniculatus bairdii and Calomys callosus. Amino acid sequences are proposed from homologous alignment with the respective sequences of white mouse and a microtine ‘stem’ developed from previous studies. These two hemoglobins differed at 11 α- and 17 β-positions, showing only one common position beyond the cricetid stems (Hesperomyini, Cricetinae) and only three common positions with the microtinae beyond the muroid or myomorph stem (Cricetidae).

Conformational relevance of the beta6Glu replaced by Val mutation in the beta subunits and in the beta(1-55) and beta(1-30) peptides of hemoglobin S.

The beta subunits of hemoglobin S showed a higher ellipticity than the beta subunits of the hemoglobin A, both in the Soret and near-ultraviolet regions. The apoderivatives of the beta subunits of hemoglobin S showed a lower helical content and a larger amount of beta conformation than the apoderivatives of the beta subunits of hemoglobin A. The beta(1-55) peptides and beta(1-30) peptides from the beta subunits of hemoglobin A and S have been separated and analyzed. The betaS(1-55) peptide showed a higher content of beta conformation and lower amount of alpha helix when compared to the betaA(1-55) peptide. This difference was present also in different concentrations of methanol. The apoderivative from the beta subunits of hemoglobin S and the betaS(1-55) peptide aggregated with increasing ionic strength. The CD measurement showed that their secondary structure did not change upon a 10-fold dilution of the sample. Very little secondary structure was present in the betaS(1-30) peptide, and the CD spectrum was very similar to that of the betaA(1-30) peptide. No significant difference in aggregation was found between the betaS(1-30) and betaA(1-30) peptides.

A comparison of the tryptic peptides of hemoglobin from two microtine genera: Clethrionomys and Ondatra

The composition of tryptic peptides was determined for Hb (major or fast electrophoretic component) from Clethrionomys rutilus and from Ondatra zibethicus. Analysis of the separated α and β-Hb chains made possible the definition of theαT9 and βT12 peptides and the resolution of uncertainties in earlier Microtus data for peptides αT9,αT12b,βT9,βT12 and βT13. The previous postulate of two deletions in peptide βT5 is supported by the present data, and the independent dipeptide βT6 appears in the Ondatra Hb. A Microtus stem sequence is inferred and used for comparison with the new data, and cladograms are given to accomodate the extended phylogenetic progression.

α Thalassemia—Caused by Gene Deletion

The thalassemia syndromes are due to quantitative abnormalities of hemoglobin peptide synthesis. Thalassemia is manifested only by hypochromia and microcytosis, with little or no anemia in the heterozygous patient (minor form); however, it causes severe hypochromic, microcytic anemia with features of hemolysis in the homozygous patient (major form). Actually, thalassemia refers to a whole spectrum of genetically determined diseases of red blood cells (RBC). The primary defect is in the rate of hemoglobin synthesis. When the genetic anomaly depresses only β-globin chain synthesis, the disorder is termed β thalassemia. This type of thalassemia is expressed in both heterozygous and homozygous forms and comprises more than 90% of all cases. β thalassemia in homozygous patients has been known as Cooley anemia. From 5% to 10% of persons carrying the thalassemia trait have hemoglobin A₂levels affected. These patients have an abnormal synthesis of hemoglobin due to a-globin deficiency or abnormality. The

Column Chromatography of Hemoglobin Peptides

(1974). Column Chromatography of Hemoglobin Peptides. CRC Critical Reviews in Clinical Laboratory Sciences: Vol. 5, No. 1, pp. 101-106.

The coding properties of multiple tRNA for valine and leucine in hemoglobin synthesis.

Multiple species of Escherichia coli transfer RNA for valine and leucine were separated from each other by countercurrent distribution. Each transfer RNA species was charged with the corresponding radioactive amino acid and then allowed to transfer its label into hemoglobin peptides in the ribosomal system from rabbit reticulocytes. Earlier experiments had shown that the minor tRNAIIbLeu would only label one position, no. 48 of the α-hemoglobin chain under these conditions. In this paper we report the coding properties of the other separated leucine tRNAs and of multiple valine tRNAs with the hemoglobin messenger. The leucine codons in the α-chain messenger can be divided into three classes with respect to their tRNA response: (a) position 48 is recognized only by tRNAIIbLeu. (b) The majority of the leucine codons can be recognized by both the two major leucine tRNAs. (c) Some leucine codons can only be recognized by one of the major leucine tRNAs. Similarly, two classes of valine codons can be distinguished in the α-chain messenger.

Hemoglobine de lapin: une variante resultant d'un allelomorphisme et non d'une ambiguite: Hb α 29 Val → Leu

Our technic of preparative separation of two α-TIV, α-TIV Leu and α-TIV Val, from rabbit hemoglobin allowed us to described the following results. 1. 1. The relative amounts of α-TIV Leu and α-TIV Val vary non-continuously according to rabbits (0; 100; 50; 50; 100; 0). 2. 2. The biosynthesis in a cell-free system of reticulocyte containing one or two α TIV hemoglobin peptides occurs at the same relative rate. 3. 3. These findings, together with our previous conclusions about the information brought by mRNA from reticulocytes, with one α-TIV hemoglobin peptide type, to the ribosomes of reticulocytes, with another type, allow us to conclude that there are two allelomorphs rabbit hemoglobins: Hb α 29 Val and Hb α 29 Leu.