Purification of NAD+ and ATP-dependent Kinases

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.

* 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 (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.

* 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.