Preparation of Pyrenyl-Actin

 

In many experimental situations it is desirable to be able to measure the extent of actin polymerisation.  The pyrenyl fluoresence assay (Kouyama. & Mihashi, 1981) is a convenient, non-destructive method for this that is very extensively used for this.  Actin (from most species) has a reactive cysteine residue at the penultimate position 374, which can be labelled with pyreneiodoacetamide at high pH. 

 

 

N-(1-pyrene)iodoacetamide

 

 

The labelled G-actin is weakly fluorescent, but upon polymerisation the fluorescence is enhanced 20 times.  The extent of polymerisation can therefore be followed by measuring the change in fluorescence using suitable wavelengths in a spectrofluorimeter.  Typically, the actin being monitored is “doped” with a small amount of the labelled actin both to conserve this material and to avoid possible artefacts.

 

 

Method.

 

1).        Dialyze 30-80 mgs of actin against:-

 

                                                            Stock                           /litre

           

            25 mM Tris pH 8.0                  1 M                              25 mls

            0.1 M KCl                               -                                   7.46 g

            2 mM MgCl2                            1 M                              2 mls

            3 mM NaN3                             1 M                              3 mls

            0.3 mM ATP                            0.1 M                           3 mls

 

2).        Clarify a 1ml sample and read the OD290.  Dilute the remainder to 1 mg/ml (= 23.8 µM; or 0.63 OD290); calculate the total nmoles.

 

3).        Transfer to a 100ml flask with a magnetic flee.

 

4).        Make up a 10mM (MW = 385) stock solution in Dimethylformamide (DMF)

Add 4 to 7 mol pyrenyliodoacetamide (Molecular Probes) per mole of actin, while gently stirring.

 

5).        Cover with foil and leave overnight at 4oC.

 

6).        Centrifuge at low speed (JA20, 5000 rpm, 5 min) to pellet the precipitated dye.

 

7).        Transfer the cleared sup. to a 42.1 (Beckman) tube and spin at 38000 rpm for 2           hrs at 4oC.

 

8).        Homogenise the pellet in Buffer G to a concentration of about 6 mg/ml.  Briefly sonicate if you have one around (it speeds up depolymerisation).  Dialyze against buffer G for 2 days, with 2 changes of buffer.

 

Buffer G :-

 

Stock

per litre

per 100 ml of 100x

 

 

 

 

 

 

2 mM Tris pH 8.0

1 M

2 ml

20ml

0.2 mM ATP

0.1 M

2 ml

20ml

 

0.5 mM DTT

dry

77 mg

0.77g

 

0.2 mM CaCl2

0.1 M

2 ml

*

 

0.002% NaN3

dry

0.2 g

2g

 

 

9).        Centrifuge at 38000 rpm for 2 hrs at 4oC.

 

10).      Gel filter with S-300 in buffer G.

 

11).      Read the OD at 344 and 290 for each fraction.

 

12).      Calculate the concentrations:-

 

                        [pyrene, µM] = OD344 / 2.2 x 104 (M-1)

 

                        [Actin, µM] = (OD290 - (OD344 * 0.127)) / 2.66 x 104 (M-1)

 

Reference:

 

Kouyama, T. & Mihashi, K. (1981). “Fluorimetry study of N-(1-Pyrenyl)iodacetamide-labelled F-actin.” Eur.J.Biochem. 114, 33-38.