John G. Pierce

Analysis of neutral sugars by gas-liquid chromatography of alditol acetates: Application to thyrotropic hormone and other glycoproteins

Abstract A method has been developed for the quantitative determination of the neutral sugars of several glycoproteins including thyrotropic hormone, thyroglobulin, and ovalbumin by gas-liquid chromatography of alditol acetates. This approach has the advantage that only one peak for each sugar is present in the chromatograms, which is particularly valuable in the determination of trace components. The same samples are analyzed for hexosamines by ion-exchange chromatography and the combination of methods permits analyses for all sugar components other than sialic acid on one or two milligrams of glycoprotein. In addition to the fucose and mannose previously reported, highly purified bovine thyrotropin contains approximately one-half residue of galactose per molecule with the human hormone purified by similar methods relatively richer in this sugar. Trace amounts of galactose have also been found in crystalline ovalbumin preparations further purified by chromatography.

Studies on the Structure of Thyrotropin: Its Relationship to Luteinizing Hormone

Publisher Summary This chapter describes the structure of thyrotropin and its relationship to luteinizing hormone (LH). The pituitary peptide hormones were among the first compounds to yield evidence for probable evolutionary relationships among proteins and peptides with different functions—that is, oxytocin and vasopressin; the corticotropins, melanotropins, and β-lipotropin; and more recently prolactin and growth hormone. The likelihood that follicle-stimulating hormone (FSH) contains subunits was recognized by Gray, and Papkoff and Ekblad reported that the FSH chain of LH may have common properties with a subunit of FSH. The dissociation of TSH and LH into two subunits raises a number of biological questions—for example, those concerning the control of biosynthesis of individual chains and the possible effects of releasing factors.

Human thyroglobulin: Studies on the native and S-carboxymethylated protein

Abstract 1. 1. The properties of human thyroglobulin prepared from both normal thyroid glands and from subjects with non-toxic colloid goiters have been studied. No significant differences in composition were found with respect to the non-iodinated amino acids, the total amount of carbohydrate, and the kinds of sugar residues found nor were differences observed in electrophoretic and centrifugal properties. The amino acid compositions are also compared to those of sheep and of hog thyroglobulin. 2. 2. The amino acid analyses showed the presence of approximately 180 half-cystine residues per molecular weight of 660 000. Within experimental error all the half-cystine residues were found to be in disulfide linkage as shown by amino acid analyses of preprations which had been treated at pH 8.6 in 8 M urea with sodium iodoacetate with and without prior reduction by mercaptoethanol. This conclusion was supported by results of amperometric titrations. 3. 3. Evidence concerning subunit structure has been obtained by physical studies of the S-carboxymethyl derivative of reduced thyroglobulin. In the presence of sodium dodecyl sulfate at pH 9.6 this derivative undergoes dissociation and sediments as a single component having an s° 20, w of 3.2–3.4 S. The molecular weight of the dissociated species was estimated to be in the range of 110 000–125 000, thus indicating at least 5 or 6 physically identical subunits or peptide chains in the native thyroglobulin molecule.

Antibodies to reduced S-carboxymethylated alpha subunit of bovine luteinizing hormone and their application to study of the purification of gonadotropin from salmon (Oncorhynchus tshawytscha) pituitary glands.

Abstract Although antisera raised against either the β (hormone specific) subunits or the α (common) subunit of bovine thyrotropin (TSH) or luteinizing hormone (LH) show no cross reaction with partially purified salmon gonadotropin, cross reactivity is found between reduced S -carboxymethylated (RCM) salmon preparations and antisera against RCM bovine LH-α. Corresponding antisera against derivatized bovine β subunits do not cross react significantly. Further purification of salmon gonadotropin preparations by chromatography was evaluated, after their derivatization, by immunodiffusion against the anti-RCM bovine LH-α. Based on absorption to concanavalin A-Sepharose columns and subsequent elution with methyl mannoside, both the gonadotropic activity and the immunoreactivity are associated with glycoprotein(s). Successive chromatography on CM-cellulose and DEAE-cellulose resulted in three distinct fractions, two of which exhibited very weak gonadotropic activity when tested in chicks but which, after derivatization, had identical cross reactivity against anti-RCM LH-α. Both these fractions show two components on gel electrophoresis in the presence of sodium dodecyl sulfate. Their reduced S -carboxymethylated derivatives yield quite similar peptide maps in which the areas occupied by glycopeptides show staining properties very similar to those of bovine LH-α. The results demonstrate the usefulness of antisera against unfolded mammalian α subunits in the investigation of the glycoprotein hormones of lower vertebrates and strongly indicate that a peptide chain with a sequence related to the common subunit of mammalian glycoprotein hormones is also present in fish gonadotropin(s).

Bovine, human and porcine thyrotropins: Molecular weights, amino- and carboxyl-terminal studies☆

Abstract The molecular weights and end-group analyses of bovine, human, and porcine thyrotropins (TSH) are reported. Both the hormones and their S-carboxymethyl derivatives behave as physically homogeneous materials during sedimentation equilibrium runs in 6 m guanidine hydrochloride. The weight average molecular weights of all three thyrotropins are approximately 25,000. No evidence was found for separation into peptide chains of smaller molecular size after disulfide bond cleavage. In the absence of a dissociating agent bovine TSH exhibited concentration-dependent aggregation. End-group analyses were in agreement with the above and showed phenylalanine to be the major amino-terminal residue in both bovine and porcine thyrotropins. In human TSH, phenylalanine and valine were present in equal proportions. In addition, 0.1 to 0.2 moles of amino-terminal aspartic acid per mole of hormone was found in each case. The carboxyl-terminal sequences of bovine and human TSH are most probably -His-Tyr-Lys-Ser-Tyr-COOH; that of porcine TSH, -His-Tyr-Lys-Tyr-Ser-COOH. About 0.2 moles of methionine and leucine which could not be accommodated in the above sequences were also found at or near the carboxyl-termini of bovine and human TSH respectively. The amino acid and carbohydrate compositions of human and porcine TSH are compared with those of bovine TSH and purified human and bovine luteinizing hormones.

The amino acid sequence of a tryptic glycopeptide from human thyroglobulin

1. 1. The glycopeptides obtained by tryptic digestion of “desialized”, S-carboxymethyl human thyroglobulin have been studied. A glycopeptide which represents a major portion of the mannose-glucosamine units has been purified and its amino acid sequence was determined to be Ala-Leu-Glu-Asp-Ala-Thr-Arg. The carbohydrate is most probably linked through the aspartic acid (asparagine) residue. The carbohydrate moiety is about twice the size reported for the corresponding oligosaccharide from calf thyroglobulin. It consists, within experimental error, of 9 residues of mannose and 2 of glucosamine. 2. 2. Evidence is presented that the more complex carbohydrate units consisting of mannose, galactose, fucose, glucosamine and a sialic acid are linked to at least two different sequences in the peptide chains of thyroglobulin. 3. 3. Galactosamine, which has not previously been detected in thyroglobulin, was also found in some fractions of the more complex type.

Partial reduction of disulfide bonds in the hormone-specific subunits of TSH and LH☆

Abstract Partial reduction at pH 7.0 of the hormone specific (β) subunit of either bovine thyrotropin or luteinizing hormone with dithioerythritol results primarily in the opening of a single disulfide bridge. The partially reduced subunits were alkylated with [1- 14 C] iodoacetic acid, followed by complete reduction and alkylation with non-radioactive iodoacetic acid. Isolation and degradation of the radioactive tryptic peptides shows that the bond primarily reduced in each β subunit links analogous half-cystine residues in the two sequences (88–95 in TSH-β and 93–100 in LH-β). These results are the first direct evidence of similar disulfide structures in hormone specific subunits of glycoprotein hormones.

The characterization of proteins by electrodialysis in starch gels

Abstract A micromethod for electrodialysis in starch gels is described in which cellophane membrane are implanted in the gels in the paths of the migrating proteins. A series of membranes of graded porosities which are suitable for studies with proteins in the molecular-weight range 10,000–100,000 have been prepared by treatment of cellophane with zinc chloride. The behavior of each protein towards an increasingly porous series of membranes varies from a situation of complete retention to one of unretarded passage and the membrane porosities at which these situations occur are related primarily to the molecular size of the protein. The technique is thus useful for approximation of molecular weight, and for studies of homogeneity and association-dissociation relationships.

Studies on the reduction and reoxidation of the disulfide bonds of the alpha and beta subunits of human choriogonadotropin.

Reoxidation of the disulfide bonds of the alpha-subunit of human choriogonadotropin after their complete reduction yields a product which is indistinguishable from the native subunit in its electrophoretic pattern in polyacrylamide gel and in its ability to recombine with the beta subunit of bovine lutropin. The circular dichroism of reoxidized human choriogonadotropin-alpha is essentially identical to that of the native alpha-subunit, except for slightly more negative ellipticity in the region of 240 mm. Hybrid hormone preparations obtained by recombination of reoxidized or native human choriogonadotropin-alpha with native lutropin-beta exhibit identical electrophoretic patterns in polyacrylamide gels, elution profiles in gel filtration, receptor binding activities, and CD spectra. However, reoxidation of human choriogonadotropin-beta under the same conditions does not yield a product which resembles the native beta subunit in its electrophoretic pattern on gels, its CD spectrum or its ability to recombine with the alpha subunit.

A study of the polymorphism of bovine thyrotropin preparations by immunochemistry and peptide mapping

Abstract Immunoelectrophoresis of highly purified bovine thyroid-stimulating hormone (TSH) preparations revealed two related antigenic components. One is inactive TSH, which also can be formed from active hormone by heat denaturation. The other and major component is the hormone. This conclusion is based on experiments in which antisera previously absorbed with inactivated TSH still neutralized the hormonal activity and on the finding that the ratio of antiserum to TSH which led to precipitation of approximately one-half the antigen also led to partial neutralization in hormone assays. Furthermore the active components observed in starch-gel electrophoresis gave identical precipitin reactions with antisera to TSH preparations which consisted either of several or one of the active components. The antibovine TSH antiserum did not show significant cross reaction with other bovine pituitary glycoproteins but did cross react with ovine and porcine TSH preparations. As previously shown by others, the bovine antibody neutralized human TSH preparations; in the present study a precipitin reaction, not identical to that obtained with ovine and bovine TSH, was also demonstrated. Immunoelectrophoresis in starch gels showed the polymorphic character of TSH even when obtained from a single anterior lobe with a minimum of manipulative procedures. Peptide maps of radioiodinated partial hydrolyzates of several of the individual active components also showed them to be essentially identical. This mapping procedure, which requires only micrograms of protein, clearly distinguished the TSH from other pituitary glycoproteins with closely related properties.