Performing a Purification of NADP+-dependent dehydrogenases and other enzymes with affinity for NADP+ Using 2’5’ ADP Sepharose 4B and Red Sepharose CL-6B

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

NADP+-dependent dehydrogenases interact strongly with 2’5’ ADP. Selective elution with gradients of NAD+ or NADP+ has allowed the resolution of complex mixtures of dehydrogenase isoenzymes using 2’5’ ADP Sepharose 4B.

Synthesis of the medium takes place in several steps. Diaminohexane is linked to 2’5’ ADP via the N6 of the purine ring. The derivatized ADP is then coupled to Sepharose 4B via the aminohexane spacer. Figure 44 shows the partial structure of 2’5’ ADP Sepharose 4B.

Partial structure of 2’5’ ADP Sepharose 4B

Fig. 44. Partial structure of 2’5’ ADP Sepharose 4B.

NADP+-dependent dehydrogenases are also members of a larger group of proteins that will interact with Procion™ Red, a synthetic polycyclic dye that shows certain structural similarities to naturally occurring NADP+. When used as an affinity ligand attached to Sepharose CL-6B, Procion Red HE-3B 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.

Partial structure of Red Sepharose CL-6B

Fig. 45. Partial structure of Red Sepharose CL-6B.

Purification Options

  Binding capacity/ml medium Maximum operating flow Comments
2’5’ ADP Sepharose 4B Glucose-6-phosphate, dehydrogenase, 0.4 mg (0.1 M Tris-HCl, 5 mM EDTA, 1 mM 2-mercaptoethanol buffer, pH 7.6).  75 cm/h* High specificity for proteins with affinity for NADP+. Supplied as a freeze-dried powder, rehydration required.
Red Sepharose CL-6B Rabbit lactate dehydrogenase, 2 mg 150 cm/h* General specificity for proteins with affinity for NADP+ and other proteins that react less specifically. Supplied as dry powder, rehydration required.

 

2’5’ ADP Sepharose 4B

Purification Example

Figure 46 shows a linear gradient elution used for the initial separation of NADP+-dependent enzymes from a crude extract of Candida utilis.

Gradient elution with 0–0.6 mM NADP+

Fig. 46. Gradient elution with 0–0.6 mM NADP+. A: non-interacting protein, B: glucose-6-phosphate dehydrogenase, C: glutamate dehydrogenase, D: glutathione reductase, E: 6-phosphogluconate dehydrogenase. (Brodelius et al., Eur. J. Biochem. 47, 81–89 (1974)).

Performing a Separation

Swell the required amount of powder for 15 min. in 0.1 M phosphate buffer, pH 7.3 (100 ml per gram dry powder) and wash on a sintered glass filter (porosity G3). 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 low and high pH wash buffers.

Cleaning

Wash 3 times with 2–3 column volumes of buffers, alternating between high pH (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) and low pH (0.1 M sodium acetate, 0.5 M NaCl, 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
2’5’ ADP Sepharose 4B 2 µmoles/ml N6-(6-aminohexyl)adenosine 2’5’ bis-phosphate coupled to Sepharose 4B by 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.

** Coupling via the N6 position of the NADP+-analogue, adenosine 2’5’ bisphosphate, gives a ligand that is stereochemically acceptable to most NADP+-dependent 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.

Red Sepharose CL-6B

Performing a separation

Swell the required amount of powder for 15 min. and wash with distilled water on a sintered glass filter (porosity G3). Use 200 ml water for each gram of dry powder, adding in several aliquots. One gram of freeze-dried material gives a final volume of approximately 4 ml. Pack a column (see Appendix 3, Column packing and preparation).

Binding buffer: Use a buffer at around neutral pH since proteins bind specifically to Red Sepharose CL-6B at this pH.

The binding capacity will depend upon parameters such as sample concentration, flow rate, pH, buffer composition and temperature. To obtain optimal purification with respect to  capacity, determine the binding capacity over a range of different pH and flow rates.

Elution buffers: use low concentrations of the free cofactor, NAD+ or NADP+ (up to 20 mM), or increase ionic strength up to 2 M NaCl or 1 M KCl.

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

Cleaning

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

Remove denatured proteins or lipids by washing with 2 column volumes of 6 M guanidine hydrochloride or 8 M urea. Alternatively, wash the column with 2 column volumes of detergent in a basic or acidic solution, e.g. 0.1% Triton X-100 in 1 M acetic acid. Remove residual detergent by washing with 5 column volumes of 70% ethanol. In both cases wash immediately with 5 column volumes of binding buffer.

Media Characteristics

  Ligand and density Composition pH stability* Mean particle size
Red Sepharose CL-6B Procion Red HE 3B 2 µmoles/ml Ligand coupled to Sepharose CL-6B using  the triazine method. Short term 3–13 Long term 4–12 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.

Chemical Stability

Stable with all commonly used aqueous buffers and additives such as 8 M urea and 6 M guanidine hydrochloride.

Storage

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

Wash media and columns with 20% ethanol in 0.1 M KH2PO4, pH 8.0 (use approximately  5 column volumes for packed media) and store at +4 to +8 °C.

Materials

     

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