Darrell N. Ward

Chromatography of luteinizing hormone from sheep pituitary glands

Luteinizing hormone has been chromatographed on carboxymethyl cellulose and hydroxylapatite ion exchange agents. The carboxymethyl cellulose resolves two fractions with luteinizing hormone activity designated LH1 and Lh2 in order of their emergence from the column. Re-chromatography demonstrated that LH2 behaved as a single component, but LH1 during processing was partially dissociated into LH2 plus a fast-moving component not retained by the ion exchange column as determined by chromatography and bioassay. The molecular weight of LH2 was estimated at 28,000 from ultracentrifuge and viscosity data. Glucosamine was present in LH1 (7.2%) and LH2 (8.0%). Bioassays indicated that both LH1 and LH2 were not significantly different in potency from the Armour LH 227-80 with respect to behaviour in the hypophysectomized rat; LH1 was less potent in the intact rat.

Comparative studies of luteinizing hormone from beef, pork, and sheep pituitaries: I. Purification and physical properties

Abstract Purification of beef, sheep, and pork luteinizing hormone (LH) from the pituitary gland is described. The fractionation has been selected to give high yield (over 80%) at each step, to obtain material for physical and chemical comparisons. Highly purified preparations have been obtained which are not completely homogeneous, however. The preparations are similar in molecular size (30,000–34,000) as judged by sedimentation-viscosity studies. All show altered sedimentation behavior at low pH, although pork LH behaves differently from sheep and beef LH. Pork LH also has approximately one-half the specific activity of the beef and sheep LH. Peptide maps of tryptic and chymotryptic digests reveal structural similarities in the preparations although some differences are apparent.

GEL FILTRATION OF PROTEINS, WITH PARTICULAR REFERENCE TO THE GLYCOPROTEIN, LUTEINIZING HORMONE.

Abstract Results are reported which show that luteinizing hormone (LH) at pH 1.3 undergoes a marked change in its physical dimensions as studied by comparison with the gel filtration behavior of a series of simple proteins and two other glycoproteins on Sephadex G-75 at pH 1.3 and pH 7.5. Comparative data for Bio-Gel P-100 at pH 7.5 is included. The calibration of the gel filtration columns is not suitable for estimation of molecular weight of glycoproteins with the limited number of glycoproteins available for reference calibration.

Semiautomatic analysis of glucosamine and galactosamine in protein hydrolyzates

Abstract The quantitative aspects of glucosamine and galactosamine analysis using the Beckman/Spinco amino acid analyzer have been described for four different chromatogrphic systems: two systems involving elution from the 150-cm column, and one each using elution from an 18-cm or 50-cm column. The procedures gave recoveries of 100 ± 2% for each hexosamine. Procedures are described whereby amino acid analysis of glycoproteins can be performed without glucosamine interference in the measurement of tyrosine and phenylalanine.

The carbohydrate components of ovine luteinizing hormone

Abstract The quantitative analysis of the carbohydrate components of ovine luteinizing hormone demonstrated the presence of 8 residues of N -acetylglucosamine, 3 residues of N -acetylgalactosamine, 7 residues of mannose, 2 residues of galactose, 1 residue of fucose and 0–1 residue of sialic acid per molecule. The number of residues of each carbohydrate subunit was calculated assuming a molecular weight of 28 000.

Amino acid composition and electrophoretic properties of ovine luteinizing hormone

Abstract The amino acid composition of ovine luteinizing hormone has been determined. Electrophoretic mobility in several buffers was studied by free boundary electrophoresis. The relatively poor buffering capacity of the protein in the neighborhood of the isoelectric point indicated by the titration curve, presented herein, and amino acid composition were used to explain the shift in isoelectric point when determined in acetate, borate, or phosphate buffers as compared to other monovalent buffers.

FURTHER PURIFICATION OF TSH-RELEASING FACTOR (TRF) FROM SHEEP HYPOTHALAMIC TISSUES, WITH OBSERVATIONS ON THE AMINO ACID COMPOSITION.

Summary Eighty thousand hypothalamus fragments of sheep brains (wet weight 55 kg) were extracted with 2 N acetic acid. The extract was filtered on Sephadex G25 in pyridine acetate 0.1 M, pH 5.6. The TRF-active zone was located by bioassay and further purified by CCD (250 transfers followed by 1,000 transfers) using the system n-butanol-pyridine-acetic acid (5:3:11), ion exchange on IRC-50 equilibrated with ammonium acetate 0.02 M, pH 5.0, and finally thin-layer chromatography on cellulose (butanol-acetic acid-water, 4:1:5). The material obtained (ca 400 μg) at the last stage of purification was active in vivo to stimulate release of TSH at ≤ 0.1 μg dry weight. Highly purified TRF has no LH- or ACTH-releasing activity, Amino acids present in this preparation are reported.

Quantitative determination of neutral monosaccharide residues in glycoproteins and glycopeptides by thin-layer densitometry

A procedure is described for the microdetermination of fucose, galactose, and mannose residues in glycoproteins and glycopeptides by direct densitometry of thin-layer chromatograms. A column of ion-exchange resin is employed for the removal of peptides, amino acids, amino sugars, and inorganic ions from hydrolyzates prior to chromatography. Separation is carried out on 250 μ cellulose layers, and aniline phthalate reagent is used for color development. Accuracy is about ±10% for a single determination. Application of the method to the analysis of human α1 acid glycoprotein and ovine luteinizing hormone is described.

Separation of the subunits of ovine luteinizing hormone by a chromatographic procedure and comparison with a countercurrent distribution procedure

Abstract 1. 1. A chromatographic procedure is described for separating ovine luteinizing hormone into two dissimilar subunits. Preliminary preparation of the S-sulfonated derivative is required. 2. 2. Analytical data are presented showing amino acid, hexosamine, and neutral sugar composition of the separated subunits, and their composition is compared with that of subunits separated by the countercurrent distribution procedure of Papkoff and Samy 12 . 3. 3. COOH-terminal analyses were performed to aid in characterizing the subunits.

Comparative studies of luteinizing hormone from beef, pork, and sheep pituitaries: II. The C-terminal amino acids

Abstract The C-terminal amino acids of beef and sheep LH have been determined by hydrazinolysis and carboxypeptidase digestion. Each LH contains two polypeptide chains, one terminating in C-terminal aspartic acid (A-chain) and one terminating in C-terminal tyrosylserine (S-chain). Although pork LH also appears to contain a C-terminal tyrosylserine, the best preparations we have obtained do not appear to be sufficiently homogeneous to make a final conclusion. From these studies, C-terminal data appear valuable for the evaluation of purity of an LH preparation.