Haptoglobins are serum glycoproteins which form complexes with hemoglobin. Three phenotypes of haptoglobin exist in serum (Hp 1-1, Hp 2-1, Hp 2-2). The latter two types exist as a series of polymers while the former type exists as a homogeneous protein. All three haptoglobin types consist of β (heavy), chains and α (light) chains which are attached by disulphides. The haptoglobin types differ in their a chains; Hp 1-1 contains only α¹ chains, while Hp 2-2 contains only α² chains and Hp 2-1 contains α¹ and α² chains. The hemoglobin-haptoglobin 1-1 complex consists of one molecule of hemoglobin attached to one molecule of haptoglobin. The thesis has been divided into three parts. The first part (Section III) is concerned with the reaction of haptoglobin with an octameric (double) hemoglobin obtained from an inbred strain of mice. In this hemoglobin each of the hemoglobin dimers is joined together by a disulphide bond. The fact that haptoglobin binds αβ dimers indicates that it is a bivalent molecule like the antibody molecule, immunoglobulin G (IgG). This bivalence and resultant resemblance to IgG is examined by studying the reaction of haptoglobin with this mouse hemoglobin in which the αβ dimer is held together by a disulphide bond. The results of both precipitation studies and acrylamide gel electrophoresis confirm the postulated bivalence of haptoglobin and its resemblance to an antibody. The second part (Section IV) of the thesis is concerned with confirming the results obtained in studying the disulphides of haptoglobin which were obtained by the cysteic acid diagonal technique. These results predicted a model in which the two halves of the haptoglobin molecule were held together by a disulphide bond at position 21α. Also the results predicted an intrachain loop disulphide between half-cystines at positions 35 and 69 in the haptoglobin α chain and an interchain disulphide between a half-cystine at position 73α and the β chain. This structure has been confirmed by studies on a cyanogen bromide fragment isolated from haptoglobin which contains the intact a chain. Also the structure has been confirmed by studies on a haptoglobin derivative in which the molecule has been split in half by the breaking of a disulphide bond. The third part (Section V) of this thesis is an investigation into the nature of the β93 sulphydryl of hemoglobin when hemoglobin is bound by haptoglobin. The results demonstrate that there is a definite change in the environment of this sulphydryl upon formation of the hemoglobin-haptoglobin complex. Studies with (14)C-iodoacetamide demonstrate however that β93 can still react in the HbHp complex. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Haptoglobins are a group of glycoproteins found in normal human serum and are capable of binding hemoglobin both in vivo and in vitro. Haptoglobin 2-1, one of the common haptoglobin phenotypes was isolated in large quantity from the ascites fluid. Upon reduction and alkylation in the presence of 8 M urea, haptoglobin dissociates into two different classes of polypeptide chains, the α and the β chains. The haptoglobin β chain, which is the larger and the only component that contains carbohydrate has been studied in the present investigation. The homogeneity of this polypeptide chain was examined using different criteria, such as DEAE-cellulose chromatography, gel filtration chromatography, urea starch gel and SDS disc gel electrophoresis. The β chain was found to be homogeneous as judged by the above different criteria. The amino acid composition and carbohydrate composition as well as the N-terminal amino acid were determined. The β chain isolated in the -S-methyl-carboxyamide form was extensively aggregated. However, upon chemical modification using succinic anhydride, the modified β chain was completely disaggregated, and it was possible to determine the accurate molecular weight of the β chain using this succinyl β chain. In order to elucidate the primary structure of the β chain, a cyanogen bromide reaction, which cleaved selectively at the methionine residues was performed. Though this cleavage was incomplete in β chain, three fragments were isolated and characterized. The amino acid sequence of the N-terminal region, the α, β interchain disulfide region and the carbohydrate attachment site region were determined. The alignment of some of the cyanogen bromide cleavage fragments were achieved using the methionine peptides from a tryptic digest of the β chain to overlap these fragments. The methionine peptides were isolated by a selective diagonal paper electrophoretic technique. The linkage between the protein and the carbohydrate in haptoglobin was established to be an aspartamido-glycosyl linkage This was demonstrated by the resistance of the linkage under mild alkali treatment and the actual isolation of an aspartic-carbo-hydrate complex. A different approach was taken to determine the primary structure of the β chain. The β chain was maleylated at its e-amino lysyl groups with maleic anhydride and later digested with trypsin. The maleylated fragments were isolated and partially characterized. Furthermore, five groups of arginine peptides were isolated. An overall structure of the β chain was deduced from the studies of the cyanogen bromide peptides, methionine peptides, tryptic maleylated peptides and the arginine peptides. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Haptoglobin is a genetically polymorphic plasma protein which possesses the property of combining specifically with hemoglobin. Haptoglobin binds hemoglobin stoichiometrically in a stable and essentially irreversible complex. The present studies are concerned with the nature of the hemoglobin combining site on haptoglobin in relation to its function. Large quantities of homogeneous haptoglobin have been prepared from ascites fluid by a newly developed method involving first, ammonium sulfate precipitation, followed by DEAE-cellulose chromatography and finally purification by Sephadex G-200 gel filtration. A Sephadex G-200 assay which separates the free hemoglobin from the Hb-Hp complex and enables direct measurement of the amount of hemoglobin bound to haptoglobin has been developed. In order to determine whether the site of polymerization of haptoglobin polymers is distinct from the hemoglobin binding site, haptoglobin polymers partially resolved on Sephadex G-200, were examined for their ability to bind hemoglobin both by the peroxidase assay and the Sephadex assay. Localization of the hemoglobin binding site on the component haptoglobin chains was achieved by determination of the ability of isolated a¹, a² and β chains to bind hemoglobin by means of the Sephadex assay. The binding of globin, myoglobin and various vertebrate hemoglobins were also examined by the Sephadex assay. The effects of environmental factors on the combination of haptoglobin with hemoglobin has been studied by alteration of ionic strength and of pH and by addition of phenol as a tyrosine analogue. The involvement of amino groups of haptoglobin in binding with hemoglobin has been studied by selective chemical modification of haptoglobin amino groups with three reagents of increasing severity followed by measurement of the ability of the chemically modified proteins to bind hemoglobin. An approximate determination of the area of the binding site in the Hb-Hp complex has been made by comparison of the extent of guanidination of amino groups of the individual components with that of the complex and also by comparison with the extent of modification in dissociated hemoglobin and haptoglobin from the guanidinated complex. The results of these studies show that the hemoglobin combining site is distinct from the site of polymerization and that the β haptoglobin chain carries the hemoglobin binding site. Haptoglobin combines with globin but not with myoglobin and it binds stoichiometrically with all animal hemoglobins examined except those of the frog and fish, which may be the result of a conformational change in the Hb-Hp complex which causes expulsion of the heme group. It is shown that electrostatic forces cannot be the sole interrnolecular forces involved in the binding. Chemical modification studies show that the area of contact between the hemoglobin and the haptoglobin in the complex involves only a small area of the haptoglobin molecule of else the area is particularly deficient in lysyl-side chains. It is also shown that amino groups are not directly involved in the binding site and that acylation of amino groups particularly with succinyl group cause a profound change in the haptoglobin molecule and its ability to bind hemoglobin is very much reduced. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (M.D.)--Københavns universitet, 1960. / Summary in Danish. Includes bibliographical references (p. 108-116) and index.

Thesis (M.D.)--Københavns universitet, 1960. / Summary in Danish. Includes bibliographical references (p. 108-116) and index.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 57 pages. Includes Vita. Includes bibliographical references.

Orientador: Lizandra Amoroso / Resumo: A urolitíase é enfermidade com importância econômica para a ovinocultura, que causa a saída prematura de ovinos machos destinados à reprodução, custos com tratamento e morte. A associação entre avaliação hemogasométrica, pesquisa de proteínas de fase aguda (PFA) como biomarcadores precoces e o estudo morfométrico do trato urinário pode ser empregada como método de auxílio diagnóstico precoce para a doença. Com esse objetivo, foram utilizados 14 ovinos hígidos, machos (não castrados), da raça Santa Inês com idade aproximada de 90 dias. Os ovinos receberam dieta experimental hiperfosfórica durante todo o período experimental, foram examinados semanalmente e após desenvolvimento da urolitíase, reorganizados em dois grupos experimentais distintos D1 (sem urolitíase) e D2 (com urolitíase) para análise comparada dos dados. No período pré-experimental e no dia do abate foram coletadas amostras de sangue venoso para avaliação hemogasométrica. Para mensurar as imunoglobulinas (A e G) e as PFA, analisaram-se as amostras dos ovinos que desenvolveram a urolitíase (D2). As coletas de sangue foram realizadas semanalmente até a manifestação clínica da enfermidade, totalizando 16 amostras. Ao término do experimento foi realizado o abate e necropsia dos ovinos, para descrição das alterações patológicas e a análise morfométrica. Fragmentos do trato urinário e fígado foram coletados e submetidos à rotina histológica e as lâminas histológicas foram descritas, seguindo-se a histomorfometria. Embo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Urolithiasis is an economically important disease for male sheep production that causes the premature calving of sheep for breeding, treatment costs and death. The association between hemogasometric evaluation, acute phase protein (APP) research as early biomarkers and urinary tract morphometric study can be employed as an early diagnostic aid for the disease. For this purpose, 14 healthy male (non-castrated) Santa Inês sheep, approximately 90 days old, were used. Sheep received a hyperphosphoric experimental diet throughout the experimental period, were examined weekly and after development of urolithiasis, reorganized into two distinct experimental groups D1 (without urolithiasis) and D2 (with urolithiasis) for comparative analysis of the data. In the pre-experimental period and on the day of slaughter, venous blood samples were collected for hemogasometric evaluation. To measure immunoglobulins (A and G) and APP, samples from sheep that developed urolithiasis (D2) were analyzed. Blood samples were collected weekly until the clinical manifestation of the disease, totaling 16 samples. At the end of the experiment, sheep were slaughtered and necropsied to describe the pathological changes and the morphometric analysis. Urinary tract and liver fragments were collected and submitted to histological routine and histological slides were described, followed by histomorphometry. Although blood pH was not different (P < 0.05) between groups, sheep that developed urolithiasis had com... (Complete abstract click electronic access below) / Doutor

Orientador: Maria de Fatima Sonati / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T10:57:40Z (GMT). No. of bitstreams: 1 Wobeto_VaniaPerettideAlbuquerque_M.pdf: 1559837 bytes, checksum: 9718fec2e739bfa4edba42baf4596e8f (MD5) Previous issue date: 2007 / Resumo: A haptoglobina (Hp) é uma proteína de fase aguda que possui propriedades antioxidantes e imunomodulatórias. Três principais genótipos/fenótipos (Hp1-1, Hp2-1, Hp2-2) demonstram eficiências distintas em suas atividades e estão associados com a suscetibilidade e evolução de várias doenças, inclusive o diabetes mellitus (DM). Alguns estudos sugerem que o polimorfismo da Hp pode influenciar o desenvolvimento da retinopatia diabética (RD), uma importante complicação microvascular desta doença. Com o objetivo de investigar essa associação em uma população brasileira, nós determinamos os genótipos da Hp em 317 pacientes diabéticos com no mínimo 10 anos de duração da doença. Os pacientes foram classificados em diabéticos do tipo 1 (DM-1) e do tipo 2 (DM-2), com e sem RD. As freqüências genotípicas dos diferentes grupos de pacientes e de um grupo controle consistindo de 142 indivíduos saudáveis, previamente estudado, foram comparadas. Nenhuma diferença significativa foi observada entre os três genótipos da Hp. Níveis de hemoglobina glicosilada (HbA1c), pressão arterial sistêmica sistólica (PAS), pressão arterial sistêmica diastólica (PAD) e duração do diabetes, considerados potenciais fatores de risco para o DM, foram também comparados. Novamente, nenhuma diferença significativa foi observada entre os genótipos. Assim nós concluímos que o polimorfismo da Hp não está associado com a presença da RD na população brasileira aqui estudada / Abstract: Haptoglobin (Hp) is an acute phase protein with antioxidant and immunomodulatory properties. Three main genotypes/phenotypes (Hp1-1, Hp2-1, Hp2-2) show distinct efficiencies in these activities and have been associated with susceptibility and outcome in several diseases, including diabetes mellitus (DM). It has been suggested that Hp polymorphism may influence the development of retinopathy, an important microvascular complication in DM. In order to investigate this association in a Brazilian population, we determined the Hp genotypes of 317 diabetic patients with at least 10 years of disease. The patients were classified as DM-type 1 and DM-type 2, with and without diabetic retinopathy (DR). The Hp genotype frequencies of the different patient groups and of a control group consisting of 142 healthy individuals who had previously been studied were compared. No significant differences were observed for the three Hp genotypes. Hemoglobin A1c levels, systolic blood pressure (SBP), diastolic blood pressure (DBP) and duration of diabetes, which are potential risk factors for DR, were also compared. Again no significant differences were observed for the three Hp genotypes. Thus, we conclude that this polymorphism is not associated with the presence of DR in the Brazilian population studied here / Mestrado / Ciencias Biomedicas / Doutor em Ciências Médicas

Orientador: Maria de Fatima Sonati / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T21:21:49Z (GMT). No. of bitstreams: 1 Santos_MagnunNueldoNunesdos_M.pdf: 1780393 bytes, checksum: f42ac636ab51c50a200800b4e28c33c3 (MD5) Previous issue date: 2009 / Resumo: O estresse oxidativo, particularmente no endotélio, exerce forte influência na gênese da vaso-oclusão e, assim, na evolução clínica e sobrevida de pacientes com Doenças Falciformes (DF). A fisiopatologia das DF está centralizada na propriedade de polimerização da desoxi-hemoglobina (Hb) S, mas fatores genéticos têm atuado como moduladores de suas complicações clínicas. A haptoglobina (Hp) é uma glicoproteína plasmática cuja função primordial é se ligar à Hb livre no plasma, impedindo a excreção renal de ferro e protegendo os vasos dos seus efeitos oxidativos. É ainda uma proteína de fase aguda positiva, com capacidade imunomodulatória. Dois alelos codominantes, HP1 e HP2, resultam em 3 genótipos/fenótipos principais, Hp1-1, Hp2-1 e Hp2-2, que correspondem a proteínas com características físico-químicas e funcionais distintas. Recentemente foi sugerido que o genótipo Hp2-2 tem sua freqüência diminuída com o aumento da idade de pacientes com DF e pode estar relacionado a um pior prognóstico. Experimentos in vitro têm demonstrado menor capacidade antioxidante e maior reatividade inflamatória relacionadas a esse fenótipo (Hp2-2). Assim, o objetivo do presente trabalho foi determinar, por PCR alelo-específica, os genótipos de Hp em 775 pacientes com DF (599 HbSS, 94 HbSC, 81 HbS/ß talassemia e 1 HbS/Deer Lodge), acompanhados no Hospital HEMOPE (Hemocentro de Pernambuco, região nordeste do Brasil) e subdivididos em 5 faixas etárias (de 3 meses a 5anos de idade - n = 237, de 6 a 10 anos- n = 96, de 11 a 15 anos - n = 89, de 16 a 20 anos - n = 95 e acima de 20 anos - n = 258). Os pacientes com idade superior a 20 anos foram ainda comparados a controles saudáveis (236 doadores de sangue da mesma região do país, ajustados para a proporção entre os sexos e pareados com os pacientes por idade e características étnicas predominantes); ambos os grupos foram também fracionados em faixas de 21 a 30 anos (140 pacientes e 111 controles) e superior a 30 anos de idade (118 pacientes e 125 controles). As distribuições das freqüências genotípicas de Hp não diferiram significativamente entre os cinco principais grupos de pacientes (p=0,7908), mesmo quando estes, adicionalmente, foram subdividos em faixas de idade de 3 meses a 16 anos (grupo pediátrico - n = 447) e superior a 16 anos (grupo adulto - n = 328) (p=0,7421). Quando os pacientes foram comparados ao grupo controle, as freqüências do genótipo Hp2-2 se mostraram inferiores (22,06% na média dos cinco grupos de pacientes - ou 22,48% no grupo V, versus 28,39% nos controles); nos pacientes com mais de 20 anos, ela sofreu redução com o aumento da idade, enquanto entre os controles se manteve estável (dado observado da comparação entre os subgrupos com idade de 21 a 30 anos - 25% de Hp2-2 nos pacientes versus 28,83% nos controles, e superior a 30 anos - 19,49% de Hp2-2 nos pacientes versus 28% nos controles). Estas diferenças, no entanto, não foram suficientes para que o nível de significância estatística fosse atingido (p = 0,6479 e 0,3780, respectivamente). Nossos resultados indicam uma redução nas freqüências do genótipo Hp2-2 dos pacientes em relação à população normal, intensificada com a progressão da idade a partir da vida adulta, mas também sugerem que as conseqüências eventualmente advindas de sua presença sejam, in vivo, minimizadas por mecanismos compensatórios inerentes aos organismos complexos. A casuística aqui analisada foi bastante expressiva, mas ainda não atingiu o tamanho amostral ideal para ser considerada representativa da população de pacientes com DF como um todo. Esses resultados se aplicam, então, exclusivamente ao grupo de pacientes ora estudado. Outras comparações efetuadas em busca da existência de interação entre o polimorfismo da Hp e os níveis de Hb Fetal, a presença de talassemia a (deleção -a3,7) e os haplótipos do cluster ß também não revelaram quaisquer dferenças significativas. / Abstract: Oxidative stress, particularly in the endothelium, exerts a strong influence on the genesis of sickle cell disease (SCD) vasoocclusion and, consequently, on patients' clinical evolution and survival. The pathophysiology of SCD is centered on the polymerization property of the desoxy-hemoglobin (Hb) S, but genetic factors can act as modulators of its clinical complications. Haptoglobin (Hp) is a plasma glycoprotein whose primary function is to bind to free hemoglobin, preventing excretion of iron by the kidneys and protecting blood vessels from its oxidative effects. It is also an acute phase positive protein with immunomodulatory capacity. Two codominant alleles, HP1 and HP2, result in 3 main genotypes / phenotypes, Hp1-1, Hp2-1 and Hp2-2, which correspond to proteins with different functional characteristics. Recently, it has been suggested that the Hp2-2 genotype frequency reduces with age in SCD patients and that this reduction may be associated with a worse prognosis. In vitro experiments have shown lower antioxidant capacity and higher inflammatory reactivity related to the phenotype (Hp2-2). Thus, the aim of this study was to determine, by allele-specific PCR, the Hp genotypes in 775 patients with SCD (599 HbSS, 94 HbSC, 81 HbS/ß-thalassemia and 1 HbS/ HbDeer Lodge), followed at HEMOPE (Pernambuco Blood Center, in the northeast region of Brazil), divided into 5 different age groups (3 months - 5 years - n=237, 6-10 years - n=96, 11-15 years - n=89, 16-20 years - n=95 and > 20 years - n=258). Those over 20 years of age were also compared with healthy controls (n=236, blood donors in the same country region, adjusted for the ratio between the gender and matched with patients by age and predominant ethnic characteristics); both groups were subdivided into 2 different age groups: 21 to 30 years (140 patients and 111 controls) and more than 30 years-old (118 patients and 125 controls). The distributions of Hp genotypic frequencies did not differ significantly among the five main groups of patients (p=0.7908), even when they, additionally, were divided into age groups from 3 months to 16 years (pediatric group - n= 447) and more than 16 years (adult group - n = 328) (p=0.7421). When the patients were compared to the control group, the frequencies of genotype Hp2-2 were lower (22.06% in means of five groups of patients - or 22.48% of group V versus 28.39% in controls); in patients over 20 years, this genotype was reduced with increased age, while it remained stable in controls (as seen from the comparison of the subgroups from 21 to 30 years of age - 25% of Hp2-2 in patients versus 28,83% in controls, and more than 30 years-old - 19.49% of Hp2-2 in patients versus 28% in controls). These differences, however, were not statistically significant (p = 0.6479 and 0.3780, respectively). Our results indicate a reduction in Hp2-2 genotype frequencies of patients, compared to the healthy controls, intensified with the progression of age from adulthood, but also suggest that the consequences resulting from their presence in vivo are probably minimized by compensatory mechanisms inherent in complex organisms. The population analyzed here was very expressive, but was not ideal to be considered as representative of SCD patients as a whole yet. These results apply only to the group of patients studied here. Other comparisons performed in search of the existence of interaction among the Hp polymorphism and Hb Fetal levels, a-thalassemia and cluster ß haplotypes also revealed no significant differences. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas