Characterization of chemically synthesized human relaxin by high-performance liquid chromatography

Canova-Davis, Eleanor; Baldonado, Ida P.; Teshima, Glen

doi:10.1016/s0021-9673(00)91242-1

Cited by 31 publications

(9 citation statements)

References 31 publications

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“…This difference in association behavior between these two relaxins is not strongly pH-dependent (unpublished observations). It is unlikely that the association of human relaxin is due to the particular method of producing this protein since human relaxin made by combining A and B chains that were synthesized by the solid-phase synthesis method of Merrifield (Canova-Davis et al, 1990) also selfassociates to dimers (Shire et al, 1988).…”

Section: Resultsmentioning

confidence: 99%

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism

Shire¹,

Holladay²,

Rinderknecht³

1991

Biochemistry

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The self-association properties of recombinant DNA derived human relaxin, and porcine relaxin isolated from porcine ovaries, have been studied by sedimentation equilibrium analytical ultracentrifugation and circular dichroism (CD). The human relaxin ultracentrifuge data were adequately defined by a monomer-dimer self-association model with an association constant of approximately 6 x 10(5) M-1, whereas porcine relaxin was essentially monomeric in solution. An approximate 5-fold increase in weight fraction of human relaxin monomer elicited by dilution of the protein resulted in no change in the far-UV CD spectrum at 220 nm. In contrast, after the same increase in weight fraction of monomer, the near-UV circular dichroism spectra for human relaxin exhibited a significant decrease in the amplitude for the CD bands near 277 and 284 nm. These CD bands, which may be assigned to the lone tyrosine in human relaxin, are superimposed on a broad envelope that is probably due to the three disulfide chromophores. Although both the human and porcine proteins contain two tryptophan residues, the near-UV CD spectra exhibit only a broad shoulder near 295 nm rather than the strong CD bands often found for tryptophan. Moreover, there is little change in this broad band after dilution of human relaxin to concentrations that resulted in a 4-fold increase in monomer weight fraction. These data suggest that dissociation of the human relaxin dimer to monomer is not accompanied by large overall changes in secondary structure or alteration in the average tryptophan environment, whereas there is a significant change in the tyrosine environment.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism

Shire¹,

Holladay²,

Rinderknecht³

1991

Biochemistry

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“…Shortly thereafter, quantities of human relaxin were produced by use of recombinant DNA techniques (unpublished experiments). The separate Aand B-chains were produced in Escherichia coli, purified, and combined to form recombinant human relaxin (rhRlx)1 in a manner similar to that previously described for a chemically synthesized human relaxin analogue (Canova-Davis et al, 1990). Hence, with use of this recombinant DNA derived molecule, it was now possible to characterize the hormone in detail, and especially to assign the disulfide bonds unequivocally.…”

mentioning

confidence: 99%

Use of recombinant DNA derived human relaxin to probe the structure of the native protein

Canova-Davis¹,

Kessler²,

Lee³

et al. 1991

Biochemistry

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This report describes the physical, chemical, and biological characterization of recombinant human relaxin (rhRlx) used as a probe to establish the disulfide pairing in native human relaxin. This strategy is necessary since native human relaxin is only available in the nanogram range. The relaxin molecule is composed of two nonidentical peptide chains, an A-chain 24 amino acids in length and a B-chain of 29 amino acids, linked by two disulfide bridges with an additional disulfide linkage in the A-chain. Native relaxin isolated from human corpora lutea was compared to rhRlx by reversed-phase chromatography, partial sequence analysis, mass spectroscopy, and bioassay. The potency of rhRlx was established by its ability to stimulate cAMP from primary human uterine endometrial cells. Native relaxin isolated from human corpora lutea was equipotent to chemically synthesized relaxin, which in turn was equipotent to rhRlx. A tryptic map was developed for rhRlx to confirm the complete amino acid sequence and assignment of the disulfide bonds. The three disulfide bonds (CysA10-CysA15, CysA11-CysB11, and CysA24-CysB23) were assigned by mass spectrometric analysis of the tryptic peptides and by comparison to chemically synthesized peptides disulfide linked in the two most probable configurations. In addition, the observed amino acid composition and sequence of rhRlx was in agreement with that predicted from the cDNA sequence with the exception that the A-chain amino terminal was pyroglutamic acid. The migration of rhRlx upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis was consistent with a monomeric structure, and the identity of the band was demonstrated by immunoblotting.

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“…As an example human relaxin has been shown to undergo reversible dimerization (Shire, Holladay, & Rinderknecht, 1991). When analyzed by size exclusion chromatography, essentially one peak is detected with a molecular weight at ∼6 kDa which is close to the expected monomer molecular weight of 6.5 kDa as determined by amino acid composition (Figure 3.18(a); Canovadavis, Baldonado, & Teshima, 1990). The weight average molecular weight is concentration dependent as shown by analytical ultracentrifugation (Figure 3.18(b)) with an extrapolated molecular weight near zero concentration of protein to that expected of a monomer.…”

Section: Aggregationmentioning

confidence: 62%

“…The weight average molecular weight is concentration dependent as shown by analytical ultracentrifugation (Figure 3.18(b)) with an extrapolated molecular weight near zero concentration of protein to that expected of a monomer. It has been reported that increasing the loading concentration to 2 mg/mL results in relaxin eluting on sizing chromatography as a dimer (Canovadavis et al, 1990). Thus, if one is not aware that reversible self-association is occurring, sizing chromatography may not detect association due to dilution on the column.…”

Section: Aggregationmentioning

confidence: 99%

Stability of monoclonal antibodies (mAbs)

Shire¹

2015

Monoclonal Antibodies

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Characterization of chemically synthesized human relaxin by high-performance liquid chromatography

Cited by 31 publications

References 31 publications

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism

Use of recombinant DNA derived human relaxin to probe the structure of the native protein

Stability of monoclonal antibodies (mAbs)

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