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Two Novel Bovine Somatotropin Species Generated from a Common Dehydroalanine Intermediate

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Abstract

Under stressed conditions such as prolonged exposure to high pH, the C-terminal disulfide bridge in bovine somatotropin (bST) is susceptible to a base catalyzed β-elimination reaction. This reaction converts the disulfide bond to a dehydroalanine residue with loss of a sulphur atom. Two altered species were isolated in pure form and determined to be generated from this dehydroalanine intermediate. One is a monomeric lanthionyl bST (L-bST) with a thioether linkage, and the other is an inter-molecular disulfide linked dimer containing a lysinoalanine. These two novel structures were unambiguously determined using various techniques including enzymatic digestion, amino acid sequencing and analysis, and mass spectrometry. The monomeric L-bST was demonstrated to be equipotent to normal bST in a hypox rat assay, thus showing that formation of lanthionine in place of this disulfide bond does not affect it bioactivity.

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Abbreviations

AEX:

Anion exchange chromatography

bST:

Bovine somatotropin

DTT:

Dithiothreitol

L-bST:

Lanthionyl bovine somatotropin

LA-dimer:

Lysinoalanyl dimer

MALDI:

Matrix-assisted laser desorption/ionization

MS/MS:

Tandem mass spectrometry

RP-HPLC:

Reverse phase high pressure liquid chromatography

SDS–PAGE gel:

Sodium dodecylsulfate polyacrylamide gel

TFA:

Trifluoroacetic acid

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Acknowledgments

We would like to acknowledge the following colleagues for their technical assistance and helpful discussion: Chip Hodam, Katherine Harris, Suzanne DeMarco, James Kostelc and Dennis Ruest. We also thank John Finnessy and James Triska for reviewing this manuscript.

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Authors and Affiliations

  1. Global Chemistry R&D, Monsanto, 800 N. Lindbergh Blvd, St. Louis, MO, 63167, USA

    Jacob S. Tou, Zi Yi Chen, Darrell A. Basler, Judy W. Cagney & S. Bradley Storrs

  2. Pfizer Global Research and Development, Pfizer Inc., 700 Chesterfield Village Parkway, Chesterfield, MO, 63017, USA

    Bernard N. Violand, James A. Carroll & Michael R. Schlittler

  3. Biopharmaceutical Research and Development, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN, 46285, USA

    Kamal Egodage

  4. Roswell, GA, USA

    Simon Poruthoor

  5. St. Louis, MO, USA

    Carol Lipartito

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  1. Jacob S. Tou
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  2. Bernard N. Violand
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  3. Zi Yi Chen
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  4. James A. Carroll
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  5. Michael R. Schlittler
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  7. Simon Poruthoor
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  8. Carol Lipartito
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  9. Darrell A. Basler
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  11. S. Bradley Storrs
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Correspondence to Jacob S. Tou.

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Tou, J.S., Violand, B.N., Chen, Z.Y. et al. Two Novel Bovine Somatotropin Species Generated from a Common Dehydroalanine Intermediate. Protein J 28, 87–95 (2009). https://doi.org/10.1007/s10930-009-9167-2

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  • DOI: https://doi.org/10.1007/s10930-009-9167-2

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