Purification or Removal of NADP+ Dependent Dehydrogenases and Other Enzymes with Affinity for NADP+

2’5’ ADP Sepharose 4B

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 3.18 shows the partial structure of 2’5’ ADP Sepharose 4B.

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

Fig 3.18. Partial structure of 2’5’ ADP Sepharose 4B.

Chromatography medium characteristics

Characteristics of 2’5’ ADP Sepharose 4B are shown in Table 3.21.

Table 3.21. Characteristics of 2’5’ ADP Sepharose 4B chromatography medium

  Ligand density (µmol/ml) Composition pH stability Average particle size (µm)
2’5’ ADP Sepharose 4B 2 N6-(6-aminohexyl)adenosine
2’5’ bisphosphate coupled to Sepharose 4B by CNBr method
Short term: 4 to 10
Long term: 4 to 10
 

1 Short term refers to the pH interval for regeneration, cleaning-in-place, and sanitization procedures. 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.

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

2’5’ ADP Sepharose 4B is available in chromatography media packs for packing in columns, see Table 3.22.

Table 3.22. Purification options for 2’5’ ADP Sepharose 4B

  Binding capacity/ml medium Maximum operating flow velocity (cm/h) Binding capacity Comments
2’5’ ADP Sepharose 4B Glucose-6-phosphate, dehydrogenase, 0.4 mg (100 mM Tris-HCl, 5 mM EDTA, 1 mM 2-mercaptoethanol buffer, pH 7.6) 75 Supplied as a freeze-dried powder, rehydration required.

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

Purification example

Figure 3.19 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 to 0.6 mM NADP+

Fig 3.19. Gradient elution with 0 to 0.6 mM NADP+. (A) noninteracting protein, (B) glucose-6-phosphate dehydrogenase, (C) glutamate dehydrogenase, (D) glutathione reductase, (E) 6-phosphogluconate dehydrogenase.

Performing a separation

Swell the required amount of powder for 15 min. in 100 mM 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).

Binding buffer: 10 mM phosphate, 150 mM NaCl, pH 7.3

If the protein of interest binds to the medium via ionic forces, it might 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 three times with 2 to 3 CV of buffers, alternating between high pH (100 mM Tris-HCl, 500 mM NaCl, pH 8.5) and low pH (100 mM sodium acetate, 500 mM NaCl, pH 4.5). Re-equilibrate immediately with 3 to 5 CV of binding buffer.

Remove denatured proteins or lipids by washing the column with 2 CV of detergent, for example, 0.1% Tween 20 for 1 min. Re-equilibrate immediately with 5 CV of binding buffer.

 

Chemical stability

Stable in 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 > 10.0 can cause loss of phosphate groups.

 

Storage

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

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

Materials