Hydroxylapatite Chromatography

Anne V. Broadhurst1

1 Roche Products Ltd., Welwyn Garden City, Hertfordshire
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 8.6
DOI:  10.1002/0471140864.ps0806s08
Online Posting Date:  May, 2001
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Abstract

Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low‐pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step‐gradient elution.

     
 
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Table of Contents

  • Startegic Planning
  • Basic Protocol 1: Low‐Pressure Chromatography for Protein Purification
  • Alternate Protocol 1: Simple Column Chromatography for Protein Purification Using Hydroxylapatite
  • Alternate Protocol 2: Batch Processing for Protein Purification Using Hydroxylapatite
  • Basic Protocol 2: HPLC of Proteins and Nucleic Acids Using Hydroxylapatite
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Low‐Pressure Chromatography for Protein Purification

  Materials
  • Hydroxylapatite matrix (see Table 8.6.1)
  • Loading buffer: 10 mM sodium phosphate, pH 6.8 ( appendix 2E)
  • Protein sample to be purified
  • Gradient solutions: 0.01 and 0.4 M sodium phosphate, pH 6.8 ( appendix 2E)
  • Loading buffer (see above) with 0.02% sodium azide
  • Side‐arm flask and vacuum source
  • Glass chromatography column (see ) with two adaptors or removable end piece and one adaptor (Fig. ), tubing, and optional packing reservoir (e.g., Pharmacia Biotech C or XK series)
  • Tubing clamps
  • Peristaltic pump
  • Buffer reservoirs
  • Fraction collector
  • UV monitor with 280‐nm filter, connected to a chart recorder
  • Gradient mixer

Alternate Protocol 1: Simple Column Chromatography for Protein Purification Using Hydroxylapatite

  • Step gradient solutions: e.g., 0.025, 0.05, 0.10, 0.25, and 0.4 M sodium phosphate, pH 6.8
  • Column with integral bottom sinter and airtight lid: e.g., Kontes Flex‐Column (Kontes Glass) or Bio‐Rad Econo‐Column
  • Frit to place on top of resin bed (Kontes Glass)

Alternate Protocol 2: Batch Processing for Protein Purification Using Hydroxylapatite

  • Step gradient solutions: e.g., 0.025, 0.05, 0.10, 0.25, and 0.4 M sodium phosphate, pH 6.8
  • Sintered‐glass funnel
  • Side‐arm flask and vacuum source
NOTE: Degassing of buffers is not essential for batch processing.

Basic Protocol 2: HPLC of Proteins and Nucleic Acids Using Hydroxylapatite

  Materials
  • 0.01 and 0.4 M sodium phosphate buffer, pH 6.8 ( appendix 2E; prepare using boiled H 2O)
  • Protein or nucleic acid standard solutions
  • Protein or nucleic acid test samples
  • 0.2 M sodium hydroxide
  • 0.01 M sodium phosphate, pH 6.8 ( appendix 2E) with 0.02% (w/v) sodium azide
  • 0.45‐µm filters (for buffers)
  • 0.22‐µm filters (for samples)
  • ∼7.5‐mm × 7.5‐cm HPLC column packed with suitable grade of hydroxylapatite (Table 8.6.1)
  • Guard column recommended for HPLC column used
  • HPLC apparatus equipped with pumps, gradient mixer, autosampler or injection loop, integrator, and UV monitor
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Figures

Videos

Literature Cited

Literature Cited
   Atkinson, A., Bradford, P.A., and Selmes, I.P. 1973. The large scale preparation of chromatographic grade hydroxylapatite and its application in protein separation procedures. J. Appl. Chem. Biotechnol. 23:517‐529.
   Bernardi, G., Giro, M.‐G., and Gaillard, C. 1972. Chromatography of polypeptides and proteins on hydroxyapatite columns: Some new developments. Biochim. Biophys. Acta 278:409‐420.
   Bio‐Rad Technical Bulletin 1115 US/EG, Bio‐Gel HPHT for protein and nucleic acid HPLC: New high performance hydroxyapatite column. Bio‐Rad Laboratories, Hercules, Calif.
   Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein‐dye binding. Anal. Biochem. 72:248‐254.
   Broadhurst, A.V., Roberts, N.A., Ritchie, A.J., Handa, B.K., and Kay, C. 1991. Assay of HIV‐1 proteinase: A colorimetric method using small peptide substrates. Anal. Biochem. 193:280‐286.
   de Gunzberg, J., Part, D., Guiso, N., and Veron, G. 1984. An unusual adenosine 3′, 5′‐phosphate dependent protein kinase from Dictyostelium discoideum. Biochemistry 23:3805‐3812.
   Gorbunoff, M.J. 1984a. The interaction of proteins with hydroxyapatite. 1. Role of protein charge and structure. Anal. Biochem. 136:425‐432.
   Gorbunoff, M.J. 1984b. The interaction of proteins with hydroxyapatite. 2. Role of acidic and basic groups. Anal. Biochem. 136:433‐439.
   Gorbunoff, M.J. and Timasheff, S.N. 1984. The interaction of proteins with hydroxyapatite. 3. Mechanism. Anal. Biochem. 136:440‐445.
   Juarez‐Salinas, H., Ott, G.S., Chen, J.‐C., Brooks, T.L., and Stanker, L.H. 1986. Separation of IgG idiotypes by high performance hydroxylapatite chromatography. Methods Enzymol. 131:615‐622.
   Kato, Y., Nakamura, K., and Hashimoto, T. 1987. High performance hydroxyapatite chromatography of proteins. J. Chromatogr. 398:340‐346.
   Kawasaki, T., Ikeda, K., Takahashi, S., and Kuboki, Y. 1986. Further study of hydroxylapatite high‐performance liquid chromatography using both proteins and nucleic acids, and a new technique to improve chromatographic efficiency. Eur. J. Biochem. 155:249‐257.
   Martinson, H.G. 1973. The basis of fractionation of single‐stranded nucleic acids on hydroxylapatite. Biochemistry 12:2731‐2736.
   Smith, G.J., McFarland, R.D., Reisner, H.M., and Hudson, G.S. 1984. Lymphoblastoid cell–produced immunoglobulins: Preparative purification from culture medium by hydroxylapatite chromatography. Anal. Biochem. 141:432‐436.
   Tiselius, A., Hjerten, S., and Levin, O. 1956. Protein chromatography on calcium phosphate columns. Arch. Biochem. Biophys. 65:132‐155.
Key References
   Gorbunoff, 1984a. See above.
  A series of three consecutive papers describing the basis of interaction of proteins with hydroxylapatite. Practical aspects are described, and there is extensive discussion of theoretical considerations.
   Gorbunoff, 1984b. See above.
   Gorbunoff and Timasheff, 1984. See above.
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