Burkart:HPLC CoAAssay

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Contents

Overview

Uses the HPLC to analyze the activity of your CoA biosynthetic enzymes (PanK, PPAT, DPCK), based on the characteristic HPLC shift to lower retention time on addition of negative charge to the CoA intermediate.

Materials

Stock Solutions

  • 250 mM ATP (151 mg/mL in 1M Tris-HCl pH 8)
  • 0.5 M KPO4 (pH 7.4)
  • 1M KCl
  • 2M MgCl2
  • CoA Biosynthetic Enzymes (His or MBP)
  • 200 mM KC12 (coumarin-pantetheine)

Master Mix (1 mL - enough for 33x30 uL rxns)

  • 420 uL H20
  • 400 uL 0.5M KPO4 (final conc 200 mM)
  • 80 uL 1M KCl (final conc 80 mM)
  • 80 uL 0.25M ATP (final conc 20 mM)
  • 20 uL 2M MgCl2 (centrifuge after adding - final conc 40 mM)
  • 5 uL 0.2M KC12 (final conc. 1 mM)

Procedure

For a 30 uL reaction:

  1. add 30 uL Master mix see recipe above) to 1.5 mL epi tubes (x4)
  2. to each epi add:
    • 2.5 uL H2O
    • 0.5 uL PanK + 1.5 uL H2O
    • 0.5 uL PanK + 1 uL PPAT + 1 uL H2O
    • 0.5 uL PanK + 1 uL PPAT + 1 uL DPCK
  3. incubate RT 30 min.
    • during this time turn on HPLC - equilibrate column (Burdick and Jackson OD5 w/ guard column front loaded - 25 cmx4.6 mm) with buffer
    • use method JLM_BUFA.M - uses buffer A = 25 mM KPO4 pH 6.55, buffer B = acetonitrile (NO TFA)
    • 0-5 min isocratic A, 5-20 min gradient to 45% B, 20-28 min gradient to 100% B, 28-29 min gradient to 100%A, 29-35 min 100%A1
  4. transfer rxns to HPLC tubes
    • 10 uL reactions - monitor @ 220 nm, 254 nm (adenine), 360 nm (coumarin)
    • coumarin pantetheine: 20.88 min
    • coumarin phosphopantetheine: 17.15 min
    • coumarin dephospho-CoA: 16.02 min
    • coumarin CoA: 14.68 min


Notes

  1. this is Jordan's mix - I've seen a lot off diff. reagent/enzyme conc work in this assay (see references) - key is to find one that works for you
  2. other conditions you may have luck with:
    • omit KCl in KPO4 buffer (Worthington special)
    • works fine in Tris-Cl - must include KCl
    • reaction tolerates pH 6.5-8.0 - 7.4 seems optimal - CoaE won't turnover positively charged CoA analogues (i.e. amino pantetheine) unless at pH 9 or higher
    • for prep scale (> 1 mL) chemoenzymatic CoA synthesis I have used the following conditions in the past: 400 mM Tris 7.4, 50 mM ATP, 4 mM pantetheine analogue, 20 mM KCl, 15 mM MgCl2, 60 uL/mL each CoA biosynthetic enzyme (PPAT, DPCK, PanK)
  3. always add MgCl2 last to master mix or will precipitate in neutral/basic buffered sol'n
  4. 25 mM pH 6.55 gives good peaks - traditional 0.05% TFA mobile phases can give peak broadening/tailing due to pH matching the pKa of the CoA phosphate (pKa~ 2)

References

Relevant papers and books

  1. Bibart RT, Vogel KW, and Drueckhammer DG. . pmid:11674364. PubMed HubMed [CoA-Drueckhammer]
  2. Nazi I, Koteva KP, and Wright GD. . pmid:14654051. PubMed HubMed [CoA-a]
  3. Dai M, Feng Y, and Tonge PJ. . pmid:11456558. PubMed HubMed [CoA-b]
  4. van Wyk M and Strauss E. . pmid:17220983. PubMed HubMed [CoA-c]
  5. Strauss E and Begley TP. . pmid:12372838. PubMed HubMed [CoA-d]
  6. Worthington AS and Burkart MD. . pmid:16357994. PubMed HubMed [Worthington-OBC]
  7. Meier JL and Burkart MD. . pmid:19374985. PubMed HubMed [Meier-Meth-Enz]
All Medline abstracts: PubMed HubMed

Contact

  • jmeier@ucsd.edu

or instead, discuss this protocol.


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