Human relaxin

Li, S., T.H. Nguyen, C. Schoneich, and R.T. Borchardt, Aggregation and precipitation of human relaxin induced by metal-catalyzed oxidation. Biochemistry, 1995. 34(17) 5762-72. 

Fig. 13.3. This figure provides another example of the incongruity of fossil records and molecular genealogy. Using humans as a standard, all mammals that purportedly are closely related have relaxins that are not more closely related to human relaxin than to that of skates and sharks. Since skates and sharks have been around in the Devonian period 380,000,000 years ago, the structural dissimilarity of the various relaxin molecules would suggest that the branching point between humans, horses, rats, etc., as well as sharks would have occurred 380,000,000 years ago. Conversely, the mammalian explosion is placed at about 60,000,000 years, which would force all the molecular genealogy lines to converge at a point 60,000,000 years ago, giving us a 300,000,000 years uncertainty. This difference is not trivial, it represents 3/3 of the total time of macro-organismic development.

Human relaxin was synthesized with either L-alanine or the unnatural D-alanine, substituting for the constant glycines in position 12 in the B chain, and we found that the modified molecules were just as active as the native ones. Furthermore, the constant glycine in B24 could be replaced by alanine with only minor disturbance 3 Yet only once so far has alanine been observed in a natural relaxin (hamster relaxin). From these experiments it follows that the glycines in position B12 and B24 must be constant for reasons other than functionality (Fig. 14.1). 

Buellesbach EE, Yang S, Schwabe C. The receptor-binding site of human relaxin II. J Biol Chem 1992 267 22957. 

Unemori EN, Beck LS, Lee WP, Xu Y, Siegel M, Keller G, Liggitt HD, BauerEA, Amento EP. Human relaxin decreases collagen accumulation in vivo in two rodent models of fibrosis. Journal of Investigative Dermatology 1993, 101, 280-285. 

Tryptic Maps of Relaxin and Relaxin B-chain. Digestion of the A-chain of human relaxin with trypsin can theoretically result in the release of five fragments that of the B-chain in the release of six fragments as illustrated in Table II. A typical tryptic map of relaxin B-chain is shown in Figure 2. The peptide was reduced and carboxymethylated with iodoacetic acid before enzymatic digestion. The peptide assignments were made after analysis of the peaks by amino acid hydrolysis for amino acid composition and confirmed by fast atom bombardment mass spectrometry (FAB-MS) as shown in Table IH... 

Amino Acid Composition of Human Relaxin B-chain... 

Figure 1. Reversed-phase HPLC chromatograms of human relaxin side fraction before and after reduction with dithiothreitol. The chromatography was performed on a Vydac C4 column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient from 18 to 50%.

Table IV is a summary of the information that has been compiled for human relaxin, growth hormone, and rt-PA.

Relaxin is a peptide hormone produced in the corpora lutea of pregnant mammals and is responsible for the widening of the birth canal to provide for the passage of the offspring. Human relaxin contains two peptide chains, an A chain of 24 amino acids, and a B chain of 29 amino acids, which are linked to each other by two disulfide bridges. 

The formation of the interchain disulhde link A24/B23 was achieved in 8M guanidinium chloride at pH 4.5 using monothiol-A chain and thiol-activated B chain in a molar ratio of 1 1.1 for 24 hours at 37 °C. The third disulhde bond was synthesized by oxidation with iodine in aqueous AcOH. Final deprotection of the partially protected relaxin was achieved by reducing Met-sulfoxide in aqueous TFA that contained NH4I and removed the For group with NaOH. The resulting human relaxin was purihed by reverse phase HPLC. 

Tregear GW, Du Y, Wang K, Wade J, Lambert P, Jones P, Southwell C, McDonald M, Niall H. The synthesis of human relaxin. Pept. Chem. 1984 22 13-18. 

Biillesbach EE, Schwabe C. Total synthesis of human relaxin and human relaxin derivatives by solid-phase peptide synthesis and site-directed chain combination. J. Biol. Chem. 1991 266 10754-10761. 

CipoUa, D.C. Shire, S.J. Analysis of oxidized human relaxin by reverse phase HPLC, mass spectrometry, and bioassays. In Techniques in Protein Chemistry II Academic Press New York, NY, 1991 543-555. 

Chen, S. Reed, B. Nguyen, T. Gaylord, N. Fuller, G. Mordenti, J. The pharmacokinetics and absorption of recombinant human relaxin in nonpregnant rabbits and rhesus monkeys after intravenous and intravaginal administration. Pharm. Res. 1993, 10, 223-227. 

S. Jaffe, R. Martin, M. Yalcinkaya, T. Cefalo, R. Chescheir, N. The pharmacokinetics of recombinant human relaxin in nonpregnant women after intravenous, intravaginal, and intracervical administration. Pharm. Res. 1993,10, 834-838. 

Huang X, Cheng Z, Sunga J, Unemori E, Zsebo K. Systemic administration of recombinant human relaxin (RHRLX) ameliorates the acute cyclosporine nephrotoxicity in rats.J Heart Lung Transplant 2001 20 253-... 

Li S, Patapoff TW, Nguyen TH, and Borchardt RT. Inhibitory Effect of Sugars and Polyols on the Metal-catalyzed Oxidation of Human Relaxin./P/tarro Set 1996 85 868-872. 

Aromatic Amino Acids (Histidine, Tryptophan and Tyrosine). Histidine residues are highly susceptible to oxidation, as shown for human growth hormone [65] and relaxin [66]. The resulting degradation products are aspartic acid, asparagines, and 2-oxo-histidine [65, 67]. Metal catalyzed oxidation of histidine may alter the secondary/tertiary structures of proteins. As has been demonstrated, oxidation of the human relaxin histidine which exists in an extended loop that joins two a-helices, alters the protein conformation, resulting in pH-dependent protein aggregation and precipitation [66, 68]. 

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