Preparative and analytical affinity chromatography of neurophysins on methionyl-tyrosyl-phenylalanyl-aminohexyl-agarose

Chaiken, Irwin M.

doi:10.1016/0003-2697(79)90076-9

Cited by 25 publications

(6 citation statements)

References 14 publications

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“…Neurophysin Preparation and Derivatization. Bovine neurophysins were acid extracted (Hollenberg & Hope, 1968) from either acetone-dried or freeze-dried posterior pituitary tissue (Pelfreez Biologicals, Rogers, AK) and purified by gel filtration and ion-exchange chromatography (Breslow et al, 1971) and then by affinity chromatography on an L-Met-L-Tyr-L-Phe-AH-column (Chaiken, 1979). Affinity-purified neurophysins were routinely freeze-dried and stored at -20 °C.…”

Section: Methodsmentioning

confidence: 99%

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Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity

Abercrombie¹,

Angal²,

Sequeira³

et al. 1982

Biochemistry

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Limited tryptic fragmentation of disulfide-intact bovine neurophysins I and II (NP-I and -II, respectively) has been found to cause selective disruption of both hormone binding and neurophysin self-association. Loss of binding interactions, measured as a loss of ability to stimulate retardation of 125I-labeled neurophysin on Met-Tyr-Phe-amino-butylaminoagarose, is complete within 3 h at 37 degrees C. Reverse-phase high-performance liquid chromatography (HPLC) analysis of tryptic digests of neurophysin I allows detection of two major protein products and the peptide fragment 1-8. Release of the latter N-terminal piece occurs at about the same rate as loss of binding interactions. Reverse-phase HPLC elution behavior before and after performic acid oxidation and amino acid composition of the protein products led to their identification as NP-I-(9-93) (the 9-93 sequence) and [des-19,20]NP-I-(9-93) (the 9-93 sequence with the dipeptide 19-20 missing) for the more rapidly and more slowly formed species, respectively. NP-I-(9-93), unlike intact neurophysin I, is not retarded strongly by either Met-Tyr-Phe-amino-butylaminoagarose or neurophysin II-Sepharose. In contrast, both NP-I-(9-93) and [des-19,20]NP-I-(9-93) are equally as effective as intact NP-I in binding neurophysin I antibodies. The role of amino-terminal residues in promoting hormone binding, self-association, and antigenic recognition interactions is considered.

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Section: Methodsmentioning

confidence: 99%

“…Bound protein was eluted with 0.2 M acetic acid (Chaiken, 1979). For preparation of inactive, derivatized neurophysins, fractions containing the unbound protein in ammonium acetate were collected and dialyzed against water to remove salt and then lyophilized and stored at -20 or -70 °C.…”

Section: Methodsmentioning

confidence: 99%

Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity

Abercrombie¹,

Angal²,

Sequeira³

et al. 1982

Biochemistry

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“…For the latter, protein was injected into a 0.9 X 25 cm column and equilibrated with 67 mM triethylamine phosphate buffer (pH 3.0) containing 23% acetonitrile, and BNP I1 was eluted with a 60-min 23-25% acetonitrile gradient and a flow rate of 3.2 mL/min. Finally, biologically active BNP I1 was obtained from the HPLC fraction by biospecific adsorption on a column of [(Met-Tyr-Phe-amino)hexyl]-Sepharose (Chaiken, 1979), equilibrated with 0.4 M ammonium acetate at pH 5.7, and elution with 0.2 M acetic acid.…”

Section: Methodsmentioning

confidence: 99%

“…Using the general expression in chromatography V = V, + upVs and noting that V, = V, + U~,~V~, we obtain from eq 2 v-v, = Because of the expansion in complexity of molecular interactions we expect to be treatable in quantitative affinity chromatography, we have adopted a new general notation, to replace that previously used (Chaiken, 1979 …”

Section: Theorymentioning

confidence: 99%

“…Because of the expansion in complexity of molecular interactions we expect to be treatable in quantitative affinity chromatography, we have adopted a new general notation, to replace that previously used(Chaiken, 1979; Abercrombie & Chaiken, 1975), which can be adapted for multiple interacting systems as well as simple ones.In these expressions V is the observed elution volume, Vm is the mobile phase volume, Vs is the stationary phase volume, and V0 is the elution volume for an unretarded molecule.…”

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Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrixes

Swaisgood¹,

Chaiken²

1986

Biochemistry

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Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)

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From gel filtration to biosensor technology: the development of chromatography for the characterization of protein interactions

Winzor

2000

J. Mol. Recognit.

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Preparative and analytical affinity chromatography of neurophysins on methionyl-tyrosyl-phenylalanyl-aminohexyl-agarose

Cited by 25 publications

References 14 publications

Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity

Effects of limited tryptic proteolysis of bovine neurophysins on molecular properties of hormone binding, self-association, and antigenicity

Analytical high-performance affinity chromatography: evaluation by studies of neurophysin self-association and neurophysin-peptide hormone interaction using glass matrixes

From gel filtration to biosensor technology: the development of chromatography for the characterization of protein interactions

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