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

Shire, Steven J.; Holladay, Leslie A.; Rinderknecht, Ernst

doi:10.1021/bi00245a006

Cited by 56 publications

(40 citation statements)

References 32 publications

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“…The remaining absorption profiles were considered as the best estimate for the residual blank absorption and were subtracted from the sample absorption profiles to obtain the c r values as a function of r. The standard equilibrium equation for a monomer has been used by the Beckman software. This software is based on nonlinear least squares techniques (17) and the Equilibrium/ Velocity Analysis Programs of Holladay and co-workers (18,19).…”

Section: Methodsmentioning

confidence: 99%

Biochemical Characterization and Ligand Binding Properties of Neuroglobin, a Novel Member of the Globin Family

Dewilde

Kiger

Burmester

et al. 2001

Journal of Biological Chemistry

430

625

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Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O 2 supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe 2؉ ) form, indicating a His-Fe 2؉ -His binding scheme. O 2 or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe 3؉ ) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k on ) and a slow dissociation rate (k off ) for both O 2 and CO, indicating a high intrinsic affinity for these ligands. However, because the ratelimiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O 2 and CO binding is suggested to be slow in vivo. Because of this competition, the observed O 2 affinity of recombinant human neuroglobin is average (1 torr at 37°C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37°C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E o ‫؍‬ ؊129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfidedependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.In addition to the well known hemoglobins (Hbs) 1 and myoglobins (Mbs), a third type of globin has recently been described in vertebrates that is predominantly expressed in the brain and other nerve tissues (1). These neuroglobins (NGBs) consist of single chains with 151 amino acids (M r ϭ ϳ17,000) that share only little sequence similarity with the vertebrate globins (Mb Ͻ 21%; Hb Ͻ 25%). Nevertheless, all key determinants of genuine globins are conserved (2). Although NGB was initially discovered in mouse and man, recent data show its presence in many different mammalian species as well as in fish, suggesting the universal occurrence of NGB in vertebrate brains. Nerve-specific globins have been sporadically observed in mollusc, annelid, arthropod, and nemertean species (3-5). These invertebrate nerve globins reach high local concentrations up to the millimolar range, which may be sufficient to facilitate O 2 diffusion or store O 2 that supports cell function during temporary hypoxia (5). The latter assumption is supported by the observation that the nervous function in the mollusc Tellina alternata under anoxic conditions depends on the oxygenation of a nerve globin (6, 7). However, the estimated amount of NGB in the vertebrate brain under nonpathological conditions is only in the micromolar range and thus is much lower than that of a typical invertebrate nerve globin (1). The physiological role of such lowly expressed globins is not well understood. Wittenberg (8) proposed that cytoplasmic globins at low concentrat...

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Section: Methodsmentioning

confidence: 99%

Biochemical Characterization and Ligand Binding Properties of Neuroglobin, a Novel Member of the Globin Family

Dewilde

Kiger

Burmester

et al. 2001

Journal of Biological Chemistry

430

625

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“…Materials-Recombinant human relaxin, in both the native (B29) and the extended (B33) forms, was synthesized at Genentech, Inc. (18). 1 The B33 form contained a 4-amino acid extension (Lys-Arg-Ser-Leu) at the carboxyl terminus of its B-chain (19) and was used for 32 P labeling.…”

Section: Methodsmentioning

confidence: 99%

Relaxin Binds to and Elicits a Response from Cells of the Human Monocytic Cell Line, THP-1

Parsell¹,

Mak²,

Amento³

et al. 1996

Journal of Biological Chemistry

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Relaxin is a 6-kDa peptide of the insulin family that is present at increased levels in the circulation during pregnancy. Its functions at that time are thought to include maintenance of myometrial quiescence, regulation of plasma volume, and release of neuropeptides, such as oxytocin and vasopressin. The protein also promotes connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis in various mammalian species. In this report, we provide evidence for a novel target of relaxin, the human monocytic cell line, THP-1. Relaxin bound with high affinity (K d ‫؍‬ 102 pM) to a specific receptor on THP-1 cells. Receptor density was low (ϳ275 receptors/cell), but binding of relaxin triggered intracelluar signaling events. Receptor density was not modulated by pretreatment with estrogen, progesterone, or a number of other agents known to induce differentiation of THP-1 cells. Cross-linking studies showed radiolabeled relaxin bound primarily to cell surface proteins with an apparent molecular mass of >200 kDa. Other members of the insulin-like family of proteins (insulin, insulin-like growth factors I and II, and relaxin-like factor) were unable to displace the binding of relaxin to THP-1 cells, suggesting that a distinct receptor for relaxin exists on this monocyte/macrophage cell line.Relaxin is a 6-kDa polypeptide with basic tertiary structural fold nearly identical to that of insulin (1). However, analysis of chemically synthesized relaxin derivatives has shown that the receptor-binding residues of the two proteins are discrete (2) and, although information is very limited (3, 4), the intracellular signaling events triggered when relaxin binds its receptor appear to be different from those triggered by insulin receptor binding. Consequently, it is not surprising that the physiological functions of relaxin are quite distinct from those of insulin and the other members of the insulin superfamily. Discovered in the 1920s, relaxin has been classically thought of as a "hormone of pregnancy" since it is present in the circulation at increased levels during pregnancy, when its synthesis is primarily directed by the corpus luteum. It serves to promote connective tissue remodeling, which allows cervical ripening and separation of the pelvic symphysis, and also acts to maintain quiescence of the myometrium (5). In addition, relaxin binding sites identified in specific areas of the brain (6) and heart (7) point to a role for it in cardiovascular function, perhaps in setting the critical thresholds for plasma volume in pregnancy (8). Relaxin is likely to play an important role in male reproductive functions as well. The protein is synthesized in the prostate, and has been shown, in vitro, to increase the motility and egg penetrating ability of sperm (9). Relaxin probably possesses non-reproductive functions as well. The identification of sites of relaxin mRNA synthesis, as well as relaxin binding sites, in the brain and heart of both males and females suggests that in those organs, the pro...

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“…Analysis by circular dichroism before and after dilution resulted in an approximate 5-fold increase in monomer, indicating that there was no difference in the far UV CD spectrum, whereas there were significant decreases in the intensity of the tyrosine CD band near 277 nm and the tyrosine and tryptophan CD band at 284 nm. Moreover, there was little change in the broad band at 295 nm due solely to tryptophan suggesting that the environment of the lone tyrosine rather than the two tryptophans changed upon dilution (Shire et al 1991) (Fig. 1).…”

Section: Self-association Of Small Non-glycosylated Proteinsmentioning

confidence: 98%

“…Early characterization of human relaxin by size exclusion chromatography (SEC-HPLC) suggested that the molecule exists in the monomeric form (data not shown). However, studies using sedimentation equilibrium analytical ultracentrifugation (SE-AUC) and circular dichroism (CD) clearly showed that this molecule undergoes concentration dependent self-association, which was not detected by SEC because of the dilution that occurs during the chromatography (Shire et al 1991). Analysis by circular dichroism before and after dilution resulted in an approximate 5-fold increase in monomer, indicating that there was no difference in the far UV CD spectrum, whereas there were significant decreases in the intensity of the tyrosine CD band near 277 nm and the tyrosine and tryptophan CD band at 284 nm.…”

Section: Self-association Of Small Non-glycosylated Proteinsmentioning

confidence: 99%

“…The weight fraction of human relaxin monomer estimated from the determined association constant by sedimentation equilibrium AUC of 100 (g/L) −1 is 0.12 at 0.5 mg/mL and 0.50 at 20 μg/mL. Adapted from Shire et al (1991) additional characterization studies of self-association needed to be done. Some of these glycoproteins contained a large percentage of sugar residues that posed several challenges in determining accurate molecular weight in solution by SEC-HPLC analysis (Shire 1994).…”

Section: Self-association Of Glycosylated Proteinsmentioning

confidence: 99%

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Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

Yadav¹,

Li²,

Scherer³

et al. 2013

Biophys Rev

Self Cite

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Early development of protein biotherapeutics using recombinant DNA technology involved progress in the areas of cloning, screening, expression and recovery/purification. As the biotechnology industry matured, resulting in marketed products, a greater emphasis was placed on development of formulations and delivery systems requiring a better understanding of the chemical and physical properties of newly developed protein drugs. Biophysical techniques such as analytical ultracentrifugation, dynamic and static light scattering, and circular dichroism were used to study protein-protein interactions during various stages of development of protein therapeutics. These studies included investigation of protein self-association in many of the early development projects including analysis of highly glycosylated proteins expressed in mammalian CHO cell cultures. Assessment of protein-protein interactions during development of an IgG1 monoclonal antibody that binds to IgE were important in understanding the pharmacokinetics and dosing for this important biotherapeutic used to treat severe allergic IgE-mediated asthma. These studies were extended to the investigation of monoclonal antibodyantigen interactions in human serum using the fluorescent detection system of the analytical ultracentrifuge. Analysis by sedimentation velocity analytical ultracentrifugation was also used to investigate competitive binding to monoclonal antibody targets. Recent development of high concentration protein formulations for subcutaneous administration of therapeutics posed challenges, which resulted in the use of dynamic and static light scattering, and preparative analytical ultracentrifugation to understand the self-association and rheological properties of concentrated monoclonal antibody solutions.

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

Cited by 56 publications

References 32 publications

Biochemical Characterization and Ligand Binding Properties of Neuroglobin, a Novel Member of the Globin Family

Biochemical Characterization and Ligand Binding Properties of Neuroglobin, a Novel Member of the Globin Family

Relaxin Binds to and Elicits a Response from Cells of the Human Monocytic Cell Line, THP-1

Assessment and significance of protein–protein interactions during development of protein biopharmaceuticals

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