RESUMO
Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.
Assuntos
Malato Desidrogenase/análise , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia em Agarose/métodos , Reações Cruzadas/imunologia , Citosol/enzimologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes de Protozoários/genética , Isoenzimas/análise , Isoenzimas/imunologia , Malato Desidrogenase/genética , Malato Desidrogenase/imunologia , Microcorpos/enzimologia , Microcorpos/genética , Microcorpos/imunologia , Mitocôndrias/enzimologia , Mitocôndrias/genética , Mitocôndrias/imunologia , Ácido Oxaloacético/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Filogenia , Proteínas de Protozoários/metabolismo , Coelhos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência/métodos , Trypanosoma brucei brucei/imunologiaAssuntos
Citosol/enzimologia , Escherichia coli/enzimologia , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Análise de Sequência de DNA , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Trypanosoma brucei brucei/genéticaRESUMO
The gene encoding tyrosine aminotransferase (TAT, EC 2.6.1.5) from the parasitic protozoan Trypanosoma cruzi was amplified from genomic DNA, cloned into the pET24a expression vector and functionally expressed as a C-terminally His-tagged protein in Escherichia coli BL21(DE3)pLysS. Purified recombinant TAT exhibited identical electrophoretic and enzymatic properties as the authentic enzyme from T. cruzi. Both recombinant and authentic T. cruzi TATs were highly resistant to limited tryptic cleavage and contained no disulfide bonds. Comprehensive analysis of its substrate specificity demonstrated TAT to be a broad substrate aminotransferase, with leucine, methionine as well as tyrosine, phenylalanine, tryptophan and alanine being utilized efficiently as amino donors. Valine, isoleucine and dicarboxylic amino acids served as poor substrates while polar aliphatic amino acids could not be transaminated. TAT also accepted several 2-oxoacids, including 2-oxoisocaproate and 2-oxomethiobutyrate, in addition to pyruvate, oxaloacetate and 2-oxoglutarate. The functionality of the expression system was confirmed by constructing two variants; one (Arg389) being a completely inactive enzyme; the other (Arg283) retaining its full activity, as predicted from the recently solved three-dimensional structure of T. cruzi TAT. Thus, only one of the two strictly conserved arginines which are essential for the enzymatic activity of subfamily Ialpha aspartate and aromatic aminotransferases is critical for T. cruzi's TAT activity.
Assuntos
Trypanosoma cruzi/enzimologia , Tirosina Transaminase/química , Tirosina Transaminase/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/fisiologia , Dicroísmo Circular , Escherichia coli/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mapeamento de Peptídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Espectrometria de Fluorescência , Espectrofotometria , Especificidade por Substrato/fisiologia , Trypanosoma cruzi/genética , Tripsina/metabolismo , Tirosina Transaminase/metabolismoRESUMO
Two malate dehydrogenase isoforms, named MDH1 and MDH2, have been purified to homogeneity from Trypanosoma cruzi epimastigotes. Both enzymes consist of subunits with a molecular mass close to 33 kDa; native molecular mass determination by gel filtration, however, indicated that MDH1 is a dimer, whereas MDH2 is a tetramer. Both isoforms did not cross-react immunologically. The N-termini of both MDH isoforms and several tryptic peptides of MDH1 (amounting to about one third of the complete molecule) have been sequenced by automated Edman degradation. The tryptic digests of both enzymes have also been analysed by mass spectrometry (MALDI-TOF MS). The apparent Km values in both directions of the reaction have been determined, as well as the possible inhibition by excess of the substrate oxaloacetate. The sequence data, together with the pI values and the presence or absence of oxaloacetate inhibition indicate that the dimeric MDH1 is the mitochondrial isoenzyme, whereas the tetrameric MDH2 is the glycosomal isoenzyme. No evidence was found for the presence of a cytosolic isoform.
Assuntos
Isoenzimas , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Reações Cruzadas/imunologia , Dimerização , Ponto Isoelétrico , Malato Desidrogenase/imunologia , Malato Desidrogenase/isolamento & purificação , Dados de Sequência Molecular , Análise de Sequência de Proteína , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Protozoários , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Citosol/enzimologia , Primers do DNA/genética , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Genes de Protozoários , Humanos , Cinética , Malato Desidrogenase/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
A broad specificity aminotransferase (BSAT), with high activity with both, aromatic amino acids and aspartate as substrates, was purified to homogeneity from promastigotes of Leishmania mexicana by a method involving chromatography on DEAE-cellulose, Red-120-Sepharose and Mono Q, and gel filtration on Sephacryl S-200. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, was 90 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequence of four internal peptides obtained by tryptic digestion. Two of these peptides, consisting of 49 amino acid residues in total, showed high similarity (57%) with corresponding sequences of plant aspartate aminotransferases, whereas they had only 33% identity with the aromatic aminotransferase of Escherichia coli, and 16% identity with the tyrosine aminotransferase from the related parasite Trypanosoma cruzi. The BSAT contained only one 1/2 Cys residue per monomer. The optimal pH for the enzyme reaction, with tyrosine and alpha-oxoglutarate as substrates, was 7.0. The apparent Km values for tyrosine, phenylalanine, tryptophan and glutamate, with oxaloacetate as co-substrate, were 1.3, 0.9, 0.9 and 171.8 mM, respectively; the value for aspartate with alpha-oxoglutarate as co-substrate was 2.5 mM, and that for alanine with alpha-oxoglutarate as co-substrate was 216 mM. The values for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as co-substrate, were 5.6, 0.71 and 0.12 mM, respectively. These results suggest that the enzyme is a broad-specificity aminotransferase, able to transaminate the aromatic amino acids, aspartate, and to a lower extent alanine, with high sequence similarity to aspartate aminotransferases.
Assuntos
Leishmania mexicana/enzimologia , Transaminases/isolamento & purificação , Transaminases/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Aminoácidos/metabolismo , Animais , Dimerização , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Especificidade por Substrato , Transaminases/químicaRESUMO
Tyrosine aminotransferase from Trypanosoma cruzi has been crystallized from PEG 4000 at pH 6.8. The crystals belong to the monoclinic space group P21 and have lattice constants of a = 59.1, b = 103.0, c = 77.8 A, beta = 113.1 degrees for a data set measured at 138 K. The presence of a non-crystallographic twofold axis together with a Matthews parameter Vm of 2.5 A3 Da-1 indicates that the asymmetric unit contains one dimeric molecule. The crystals diffract to at least 2.7 A and are stable in the X-ray beam in a shock-frozen state. Native data sets have been collected at temperatures of 285 and 138 K using a Siemens X1000 detector on a rotating-anode generator.
Assuntos
Trypanosoma cruzi/enzimologia , Tirosina Transaminase/química , Animais , Cristalização , Cristalografia por Raios X , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestruturaRESUMO
Tyrosine aminotransferase purified from epimastigotes of Trypanosoma cruzi displays an additional activity of alanine aminotransferase, absent in all other tyrosine aminotransferases characterized so far. Since the parasite's genome contains a high number of copies of the tyrosine aminotransferase gene, we could not rule out the possibility that two very similar proteins, with changed specificity due to a few amino acid substitutions, might be responsible for the two activities. We have now expressed in Escherichia coli a recombinant tyrosine aminotransferase as a fusion protein with glutathione S-transferase. The purified fusion protein, intact or after thrombin cleavage, displays tyrosine aminotransferase and alanine aminotransferase activities with apparent Km values similar to those for the natural enzyme, thus proving that they belong to the same protein.
Assuntos
Alanina Transaminase/genética , Trypanosoma cruzi/genética , Tirosina Transaminase/genética , Alanina Transaminase/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/genética , Glutationa Transferase/genética , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Trypanosoma cruzi/enzimologia , Tirosina Transaminase/metabolismoRESUMO
An aromatic L-alpha-hydroxyacid dehydrogenase (AHADH) was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, hydrophobic interaction chromatography on Phenyl-Sepharose and affinity chromatography on Affi-Gel Blue. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is about 80 kDa, the native enzyme is a dimer of similar subunits. The amino acid composition was determined, as well as the sequences of 4 internal peptides obtained by CNBr cleavage at Met residues, and one peptide obtained after tryptic digestion. Three of the peptides presented considerable sequence similarity with the corresponding sequences of several malate dehydrogenases. The optimal pH for the enzyme reaction with p-hydroxyphenyl pyruvate and NADH as substrates was 7.5; that for the reverse reaction was 9.5. The apparent Km values for phenylpyruvate and p-hydroxyphenyl-pyruvate were 48 and 117 microM, respectively; that for L-phenyllactate in the reverse reaction was 420 microM. The enzyme was much less active with alpha-isocaproic acid as substrate, and other acids, including pyruvic and oxaloacetic, were not substrates at all. L-phenyllactic acid, but not the D-isomer, acted as substrate. The enzyme can therefore be considered as a general aromatic L-alpha-hydroxyacid dehydrogenase. The low apparent Km value for NADH (25 microM in the presence of phenylpyruvate) makes AHADH a candidate for the reoxidation of cytosolic NADH in T. cruzi.
Assuntos
Oxirredutases do Álcool/isolamento & purificação , L-Lactato Desidrogenase , Lactato Desidrogenases , Trypanosoma cruzi/enzimologia , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Epimastigotes of Trypanosoma cruzi in culture produce and excrete into the medium small amounts of phenyllactic acid and p-hydroxyphenyllactic acids, presumably arising from the catabolism of the aromatic amino acids phenylalanine and tyrosine, respectively. This production might constitute a minor pathway for the reoxidation of cytosolic NADH, through the concerted action of tyrosine aminotransferase and aromatic alpha-hydroxyacid dehydrogenase.
Assuntos
Hidroxiácidos/metabolismo , Lactatos/metabolismo , NAD/metabolismo , Oxirredutases/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Indóis/metabolismo , Modelos Biológicos , Oxirredução , Fenilpropionatos/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Tyrosine aminotransferase was purified to homogeneity from epimastigotes of Trypanosoma cruzi by a method involving chromatography on DEAE-cellulose, gel filtration on Sephacryl S-200 and chromatography on Mono Q in an f.p.l.c. system. The purified enzyme showed a single band in SDS/PAGE, with an apparent molecular mass of 45 kDa. Since the apparent molecular mass of the native enzyme, determined by gel filtration, is 91 kDa, the native enzyme is a dimer of similar subunits. The amino-acid composition was determined, as well as the sequences of three internal peptides obtained by CNBr cleavage at Met residues. Both criteria suggest considerable similarity with the tyrosine aminotransferases from rat and from human liver. The enzyme contains nine 1/2 Cys residues, three free and the others forming three disulphide bridges. The enzyme is not N-glycosylated. The isoelectric point is 4.6-4.8. The optimal pH for the reaction of the enzyme with tyrosine as a substrate is 7.0. The apparent Km values for tyrosine, phenylalanine and tryptophan, with pyruvate as a co-substrate, were 6.8, 17.9 and 21.4 mM, respectively, whereas those for pyruvate, alpha-oxoglutarate and oxaloacetate, with tyrosine as a substrate, were 0.5, 38 and 16 mM respectively. The purified tyrosine aminotransferase acts as an alanine aminotransferase as well and the activity seems to reside in the same enzyme molecule. The results suggest that the enzyme is a general aromatic-amino-acid transaminase, with high sequence similarity to tyrosine aminotransferases from rat and human liver.
Assuntos
Trypanosoma cruzi/enzimologia , Tirosina Transaminase/isolamento & purificação , Tirosina Transaminase/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Cinética , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Homologia de Sequência de Aminoácidos , Termodinâmica , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.
Assuntos
Oxirredutases/isolamento & purificação , Fenilpropionatos/metabolismo , Trypanosoma cruzi/enzimologia , Tirosina Transaminase/isolamento & purificação , Animais , Ciclo Celular , Frações Subcelulares/enzimologia , Trypanosoma cruzi/ultraestruturaRESUMO
The effect of acetylation of tyrosine residues on the binding capacity of human growth hormone (hGH) to rat liver lactogenic and somatogenic receptors was studied. When 3.7 tyrosine and 4.8 lysine residues were acetylated with N-acetylimidazole, both the in vivo and the in vitro capacities of hGH to compete with 125I-labeled bovine growth hormone for somatogenic binding sites greatly decreased. Acetylation also affected the in vitro binding capacity to lactogenic sites. Most of the somatogenic binding activity was recovered by hydroxylamine treatment, which removes O-acetyl groups from tyrosine residues but not N-acetyl groups from lysine residues. The same treatment partially restored lactogenic binding capacity. The reactivity of hGH tyrosine residues to N-acetylimidazole, together with previous evidence, suggests that: (a) Tyrosine residues 160 and 164, when acetylated, are likely to be responsible for the low binding activity of acetylated hGH. (b) Tyrosine 160 may play a significant role in hGH interaction with lactogenic receptors.
Assuntos
Hormônio do Crescimento/metabolismo , Imidazóis/química , Fígado/química , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos , Receptores da Somatotropina/metabolismo , Tirosina/química , Acetilação , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Feminino , Hormônio do Crescimento/química , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Oxirredução , Ratos , Espectrofotometria UltravioletaRESUMO
Reactivity of arginine residues in human growth hormone was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit to a pseudo first order curve, with an apparent velocity constant k = 1.26 x 10(-2) min-1 and a maximum modification of 9.6 out of the 11 arginines of the molecule. Modification led to a decrease in binding capacity to both lactogenic and somatogenic rat liver receptors. In either case Tsou plots suggest that the modification of two arginine residues is responsible for this behavior, although it cannot be ascertained whether the two relevant residues are the same for both receptor types. Circular dichroism studies indicated no apparent changes in protein conformation in the modified hormone. Binding capacity was restored upon regeneration of arginines by incubation with Tris-HCl buffer. Only the carboxy-terminal peptide was isolated by HPLC from a tryptic digest of succinylated Arg-modified hGH, indicating that 183 is the nonreacting arginine residue.
Assuntos
Arginina/metabolismo , Cicloexanos/farmacologia , Cicloexanonas/farmacologia , Hormônio do Crescimento/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos , Receptores da Somatotropina/metabolismo , Animais , Cicloexanonas/metabolismo , Humanos , Indicadores e Reagentes , Fígado/metabolismo , Lactogênio Placentário/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
Three different methods have been applied to the prediction of secondary structure. The prediction that better fitted the chemical data was chosen. Two regions of the bovine growth hormone molecule (111-117 and 166-174) appear to be exposed to the solvent, according to hydropathic analysis but have several charged residues not reactive towards their specific reagents. Two molecular domains are postulated, each one bearing a region with charged residues on its surface and interacting with the other in the molecule by means of saline bridges. The hydrophobic core of the molecule is formed by the ensemble of the hydrophobic region predicted between residues 81 and 108, and the hydrophobic faces of the amphiphilic helices 109-127 and 9-33.
Assuntos
Hormônio do Crescimento/química , Sequência de Aminoácidos , Animais , Bovinos , Dados de Sequência Molecular , Conformação Proteica , Difração de Raios XRESUMO
The alpha-amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the alpha-amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.
Assuntos
Hormônio do Crescimento/análogos & derivados , Hormônio do Crescimento/síntese química , Prolactina/análogos & derivados , Prolactina/síntese química , Animais , Ligação Competitiva , Dicroísmo Circular , Humanos , Cinética , Fígado/metabolismo , Prolactina/metabolismo , Conformação Proteica , Receptores da Prolactina/metabolismo , Ovinos , Relação Estrutura-AtividadeRESUMO
Derivatives of bovine growth hormone, containing monoaminotyrosyl residues in positions 35, 42 and 174, were treated at pH 3.6 with a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Under these conditions aminotyrosyl groups reacted. On changing the pH to 9.3, the second fluorine atom of the reagent was substituted with the sterically adjacent side groups of lysine, since the excess of reagent had been previously removed. The modified protein underwent cyanogen bromide treatment. Peptides containing the crosslinks were purified from tryptic digests of the cyanogen bromide fragments by HPLC. Results show that aminoTyr 174 was able to form dinitrophenylene bridges with Lys 111, Lys 29 and Lys 170. AminoTry 35 was found crosslinked to Lys 29. Taking into account the size of the reagent, it may be inferred that Lys 29, 111 and 170 are located at approximately 5 A from Tyr 174 in the bovine growth hormone molecule.
Assuntos
Dinitrofluorbenzeno , Hormônio do Crescimento/análise , Nitrobenzenos , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Dinitrofluorbenzeno/análogos & derivados , Concentração de Íons de Hidrogênio , Dados de Sequência MolecularRESUMO
The bovine growth hormone dimeric form covalently stabilized by cross-linking with dimethyl suberimidate (DMS) and the hormone modified by DMS without forming covalent links with other hormone molecules (DMS-bGH) were oxidized with chloramine-T at molar-ratios of 2 and 50 with respect to methionine content. The extent of oxidation undergone by each methionine residue, estimated on the purified tryptic peptides, closely resembled that obtained for the native hormone, thus suggesting that methionine residues are not involved in the protomers interaction area. Evaluation of the reactivity of tyrosine residues toward tetranitromethane indicated that, in both the covalent dimer and DMS-bGH, tyrosine residues 35, 174 and 142 are the more susceptible to undergo reaction. Net charges can be induced in the iodotyrosine residues in the iodinated hormone, by setting the pH at 10.5. At this pH, dissociation of a fraction of uniformly iodinated hormone was observed in the derivatives containing 2 or more iodine atoms, indicating that tyrosine residues might integrate the contact area between protomers.