Performing a Purification of NAD+-dependent dehydrogenases and ATP-dependent kinases with 5’ AMP Sepharose 4B, HiTrap Blue HP and Blue Sepharose 6 Fast Flow

Extracted from Affinity Chromatography Principles and Methods, GE Healthcare, 2007

NAD+-dependent dehydrogenases and ATP-dependent kinases interact strongly with 5’ AMP so that selective elution with gradients of NAD+ or NADP+ enables the resolution of complex mixtures of dehydrogenase isoenzymes, using 5’ AMP Sepharose 4B.

Synthesis of 5’ AMP Sepharose 4B takes place in several steps. Diaminohexane is linked to AMP via the N6 of the purine ring. The derivatized AMP is then coupled to Sepharose 4B via the aminohexane spacer.

NAD+-dependent dehydrogenases and ATP-dependent kinases are also members of a larger group of proteins that will interact with Cibacron Blue F3G-A, a synthetic polycyclic dye that shows certain structural similarities to the cofactor NAD+. When used as an affinity ligand attached to Sepharose 6 Fast Flow or Sepharose HP, Cibacron Blue F3G-A will bind strongly and specifically to a wide range of proteins. Some proteins bind specifically due to their requirement for nucleotide cofactors, while others, such as albumin, lipoproteins, blood coagulation factors and interferon, bind in a less specific manner by electrostatic and/or hydrophobic interactions with the aromatic anionic ligand.

Purification Options

  Binding capacity Maximum operating flow Comments
5’ AMP Sepharose 4B Lactate dehydrogenase, 10 mg/ml medium (0.1 M phosphate buffer, pH 7.0 at +20 °C) 75 cm/h* High specificity for proteins with affinity for NAD+. Supplied as a freeze-dried powder, rehydration required.
HiTrap Blue HP Human serum albumin, 20 mg/column   Human serum albumin, 100 mg/column 4 ml/min (1 ml column)     20 ml/min (5 ml column) General specificity for proteins with affinity for NADP+ and other proteins that react less specifically. Prepacked columns.
Blue Sepharose Fast Flow Human serum albumin, 18 mg/ml medium 750 cm/h* General specificity for proteins with affinity for NADP+ and other proteins that react less specifically. Supplied as a suspension ready for column packing.

* See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

5’ AMP Sepharose 4B

Performing a Separation

Swell the required amount of powder for 15 min. in 0.1 M phosphate buffer, pH 7.0 (100 ml per gram dry powder) and wash on a sintered glass filter. Pack the column (see Appendix 3, Column packing and preparation).

Binding buffer: 10 mM phosphate, 0.15 M NaCl, pH 7.3

If the protein of interest binds to the medium via ionic forces, it may be necessary to reduce the concentration of NaCl in the binding buffer.

Elution buffers: use low concentrations of the free cofactor, NAD+ or NADP+ (up to 20 mM) with step or gradient elution.

If detergent or denaturing agents have been used during purification, these can also be used in the high and low pH wash buffers.

Cleaning

Wash 3 times with 2–3 column volumes of buffers, alternating between high pH (0.5 M NaCl, 0.1 M Tris-HCl, pH 8.5) and low pH (0.5 M NaCl, 0.1 M sodium acetate, pH 4.5). Re-equilibrate immediately with 3–5 column volumes of binding buffer.

Remove denatured proteins or lipids by washing the column with 2 column volumes of detergent e.g. 0.1% Triton X-100 for 1 minute. Re-equilibrate immediately with 5 column volumes of binding buffer.

Media Characteristics

  Ligand density Composition pH stability* Mean particle size
5’ AMP Sepharose 4B 2 µmoles/ml N6(6-aminohexyl-) 5’ AMP  coupled to Sepharose 4B  using CNBr method** Short term 4–10 Long term 4–10 90 µm

* Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

** The attachment of the ligand via an alkyl linkage to the N6 amino group gives a stable product that is conformationally acceptable to most 5’ AMP- or adenine nucleotide cofactor-requiring enzymes.

Chemical Stability

Stable to all commonly used aqueous buffers and additives such as detergents. Avoid high concentrations of EDTA, urea, guanidine hydrochloride, chaotropic salts and strong oxidizing agents. Exposure to pH >10 may cause loss of phosphate groups.

Storage

Store freeze-dried product below +8 °C under dry conditions.

Wash media and columns with 20% ethanol at neutral pH (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.

HiTrap Blue HP, Blue Sepharose 6 Fast Flow

The information supplied for the purification or removal of albumin (page 70, Purifcation or removal of albumin) is applicable also to the purification of enzymes with an affinity for NAD+.

Performing a Separation

As for albumin (see page 72, Purifcation or removal of albumin), but note the following:

For elution use low concentrations of the free cofactor, NAD+ or NADP+ (1–20 mM), or increase ionic strength (up to 2 M NaCl or KCl, 1 M is usually sufficient).

For less specifically bound proteins: use higher concentrations of cofactor or salt or more severe eluents such as urea or potassium isothiocyanate. Polarity reducing agents such as  dioxane (up to 10%) or ethylene glycol (up to 50%) may be used.

Materials

     

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