Characterization of the circulating form of porcine relaxin: biological activity and terminal amino acids.

Relaxin is structurally related to insulin, and it induces pregnancy-related changes in the reproductive tract of several mammalian species. Relaxin isolated from the ovaries of pregnant sows has been used for primary structure determination, for much of the biological characterization of the hormone, and for the development of RIAs. Immunoreactive (IR) relaxin is found in peripheral blood during pregnancy in pigs and other species, but it has not been established that the substance identified by RIA is structurally or biologically equivalent to the native ovarian hormone. IR relaxin was, therefore, isolated from peripheral plasma of late pregnant gilts (days 112-114) for bioassay and determination of terminal amino acid residues. IR relaxin was monitored by a specific homologous RIA during fractionation of plasma by gel filtration, cation exchange, and hydrophobic binding to octadecysilica. IR relaxin circulates unbound and is equipotent with ovarian relaxin in the mouse pubic ligament bioassay. Amino acids released from IR relaxin by pyroglutamic aminopeptidase and carboxypeptidase-Y were converted to their 4-dimethylamino-azo-4'-sulfonyl derivatives for identification by HPLC. The B-chain of IR relaxin had an amino-terminal pyroglutamic acid. Amino acids sequentially released from the carboxy-terminal indicated a chain length of 28-30 amino acids, suggesting a heterogeneity reminiscent of that of ovarian relaxin isolated by other methods. Arginine was released from the free amino-terminal by dimethylaminozaobenzene-isothiocyanate degradation, indicating an intact A-chain of 22 amino acids. Blood immunoreactive relaxin in pigs is, therefore, a secreted biologically active form of relaxin with an amino acid composition similar to that of the form stored in the corpus luteum.

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein.

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.

Effect of long-chain fatty acids on the binding of thyroxine and triiodothyronine to human thyroxine-binding globulin.

The effect of long-chain fatty acids on the binding of thyroxine to highly purified human thyroxine-binding globulin has been studied by equilibrium dialysis performed at pH 7.4 and 37 degrees C. At a fixed molar ratio of 2000:1 of fatty acid to thyroxine-binding globulin, the degree of binding inhibition based on the percent change in nK value relative to the control as determined from Scatchard plots was: palmitic, 0%; stearic, 0%; oleic, 76%; linoleic, 69%; and linolenic, 61%. At a 500:1 molar ratio of oleic acid to thyroxine-binding globulin, equivalent to 0.125 mM free fatty acid in serum, thyroxine binding was inhibited by 18%, increasing to 93% at a 4500:1 molar ratio. At molar ratios of oleic acid to thyroxine-binding globulin of 1000:1, 2000:1 and 4000:1, the degree of inhibition of triiodothyronine binding was 24%, 41% and 76%, respectively. The results indicate that the unsaturated long-chain fatty acids are potent inhibitors of thyroxine binding to thyroxine-binding globulin, whereas the saturated fatty acids have little or no effect on thyroxine binding.

Evaluation of N-bromoacetyl-L-thyroxine as an affinity label for the thyroxine (T4)-binding site in human T4-binding globulin.

The T4 analog N-bromoacetyl-L-T4 (BrAcT4) has been investigated as a possible affinity labeling reagent for identification of amino acids located within the T4-binding site in T4-binding globulin (TBG). As shown by fluorescence measurements involving displacement of 8-anilino-1-naphthalene-sulfonic acid from TBG, BrAcT4 is an effective competitor for the T4-binding site in TBG, with an association constant one seventh that of T4. Covalent modification of TBG by BrAcT4 was a slow process; after 48 h at a 10:1 molar ratio of [14C] BrAcT4 to TBG, incorporation of the 14C label reached 0.58 mol/mol protein or 77% of the theoretical value, correcting for 0.25 mol residually bound T4 in the original TBG sample. When [14C] BrAcT4 was reacted with TBG in the presence of T4, a partial inhibition of 25% in the degree of modification was obtained. The low inhibition of incorporation of label in the presence of T4 may be attributed to displacement of T4 from the binding site by BrAcT4 during the 20-h reaction time. To determine the effect of modification of the protein on binding activity, TBG was reacted with [14C]BrAcT4, and the binding capacity of modified TBG was determined by equilibrium dialysis. Three different TBG and three different [14C]BrAcT4 preparations were used. In two experiments, there was no reduction in binding capacity of modified TBG compared to that of control, although 0.6 and 0.48 mol label were incorporated per mol protein. In the third experiment, the decrease in binding capacity of modified TBG was 45% of the expected value. The lack of correspondence between the reduction in binding capacity and the degree of modification indicates that instead of reacting with amino acids within the T4-binding site, BrAcT4 derivatizes amino acids that are near but not actually part of the site. Covalent attachment of BrAcT4 to amino acids outside the T4-binding site places this compound in the category of an exoaffinity labeling reagent with regard to TBG and limits its usefulness for unequivocal identification of amino acids in the protein that participate directly in binding T4.

Thyroxine-protein interactions. Binding constants for interaction of thyroxine analogues with the thyroxine binding site on human thyroxine-binding globulin.

The binding constants for interaction of various thryoxine analogues with the thyroxine binding site on human thyroxine-binding globulin have been determined. Equilibrium dialysis, at pH 7.4 and 37 degrees C, was used to measure the competitive effects of different iodothyronine compounds on the binding of 125I-labeled thyroxine to highly purified thyroxine-binding globulin. Relative to L-thyroxine, K = 6 . 10(9) M-1, the association constants of some important analogues were D-thyroxine, 1.04 . 10(9) M-1, 3,5-diiodo-3'-isopropyl-L-thyronine, 4.9 . 10(8) M-1; L-triiodothyronine, 3.3 . 10(8) M-1, 3,3',5'-DL-triiodothyronine (reverse triiodothyronine), 3.1. 10(8) M-1; tetraiodothyropropionic acid, 2.7 . 10(8) M-1; tetraiodothyroacetic acid, 2.6 . 10(8) M-1; 3', 5'- diiodo-DL-thyronine, 8.3 . 10(7) M-1; and 3,5-diiodo-DL-thyronine, 7.1 . 10(7) M-1. Calculation of the deltaG0 values for binding of the analogues indicates that a major contribution to the free energy favoring binding is made by the alanine side chain of thyroxine. A change in configuration of the alpha-amino group from the L to D form causes an unfavorable change of 1 kcal/mol in the free energy of binding. Removal of the alpha-amino group as in tetraiodothyropropionic acid causes an unfavorable change of 1.9 kcal/mol in the free energy of binding. With regard to ring substituents, the results indicate that the two inner 3,5-iodines make about the same contribution to binding as the two outer 3', 5'-iodines.

Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin.

The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.