Reggie StallworthMyosin from Myofibrils
Myosin from Myofibrils
If using glycerinated myofibrils they must be washed to remove the glycerin before extracting the myosin. Use the following washing procedure:
Set-up:
Day before the prep:Prepare all buffers in flasks a day before the prep. Store in +4oC.
Reserve the centrifuges for the first 2 days of the prep and the ultracentrifuge for the second day of the prep.
If using Biuret assay to determine [protein], ensure assay solutions are prepared.
Day of the prep:Turn on centrifuge and cool down to ~0oC
Solutions:
pHChemical[Stock]Volume/L[Final]
Rigor Buffer7.0KAc 2.5M 24 ml60mM
(9RB(KAc))MgCl2 1.0M 6 ml6mM
EGTA 0.5M 2 ml1mM
NaN3 1.0M 1 ml1mM
MOPS 1.0M 25 ml25mM
Myosin Precip.7.0EDTA 0.5M 4 ml2mM
Buffer (MPB)MOPS 1.0M 20 ml20mM
Myosin Buffer7.0KCL 37.28g500mM
(MB)EDTA 0.5M 0.2 ml0.1mM
MOPS 1.0M 5 ml5mM
KCL (FS 74.56)--- solid370mM
450mM
ATP (MW 551.1)7.0 solid4mM
5mM
Make up fresh, pH with solid KOH until near 7.0.
IMPORTANT REMINDERS:
Keep protein solution on ice and buffers cold at ALL times!
Use ONLY cold H2O for solutions made throughout the prep!
If starting with ~500ml glycerinated myofibrils use 2 250ml bottles.
1.Add glycerinated myofibrils to centrifuge bottles. To 1 vol. glycerinated fibrils add 3 vol. Rigor Buffer (RB). Mix well.
2.Centrifuge at 1500g for 10 min at 0C. to remove glycerin. Discard supernatant. Repeat centrifugation until all myosin is pelleted.
3.Resuspend myosin pellet with fresh RB with small additions of RB until full (~250 ml per centrifuge tube) and thoroughly mix. Centrifuge at 1500 g for 10min at 0C. Discard supernatant. Add fresh RB and centrifuge solution @2,000 g for 20 minutes. Discard supernatant.
4.Carefully transfer pellet with spatula into a 2L beaker. Rinse centrifuge bottles out with small amounts of cold RB(KAc) and add this to the pellet. Suspend the pellets with small additions of cold RB(KAc) to 60ml (1/4 bottle) and mix well until smooth avoiding lumps.
5.Next, if using either freshly prepared or washed glycerinated myofibrils:
A.)Estimate amount of protein from [protein] (if known for starting material).
or
B.)Determine the new volume and protein concentration of the purified myofibrils (Biuret Assay)
( see Biuret Solutions and Biuret Assay)
Volume=
[mg/ml]=
6.Dilute myofibrils to 25mg/ml with cold RB (KAc). ( volume of KAc to add ([mg/ml]*volume-volume=) Measure total volume.
Volume=
7.Extract myosin by bringing the homogenate to:
0.004M ATP
0.37M KCl
NOTE: The ATP will dissociate the myosin from actin, and the high salt will solubilize the myosin.
Add the ATP first, stir for 10” and then add the KCl and stir for another 10”. Let the solution sit in the coldbox for 10’ (no longer or actin is also extracted from the myofibrils). Stir for 15” every 3’. Immediately centrifuge at 20,000g for 60 min at 0C or at 40,000g for 30 min at 0C. This removes most of the non-myosin. Discard pellet and measure volume.(V’)
Volume (V’) =
8.Pour supernatant into flask(s) containing 8 x V’ of cold MPB (a cloudy precipitate of myosin should form in the solution right away). Allow the solutions to sit overnight in coldbox so the precipitated myosin can settle to the bottom of the beakers.
Volume MPB =
NOTE: If you start from a rabbit going to myofibrils, you can get to this step in 8 hrs with 2 people working. Then it works out well to let the precipitated myosin settle overnight so there isn’t so much to centrifuge, occasionally there won’t be much settling.
THIS IS A GOOD STOPPING POINT!!!
9.Pellet the precipitated myosin the following morning by centrifuging the MPB at 10,000g for 10 min at 0C. Discard clear supernatant. ( If supernatant is not clear spin for an additional 10’)
NOTE: For large batches it will be necessary to do successive spins, adding additional solution to the pellets after discarding the supernatant.
10.Rinse the beakers with MPB to get any myosin precipitate left behind and add this to the solution in the centrifuge tubes before spinning. If the myosin precipitate has settled well at the beaker bottom, it may be possible to decant off some of the MPB, thus reducing the total volume and time required for centrifugation.
11.Carefully transfer the pellets containing precipitated myosin out of the centrifuge tubes and pool them into a clean beaker when finished centrifuging. . Rinse the centrifuge tubes with small amounts of MB(1-3ml total)to dissolve any myosin left behind. Dissolve the myosin by resuspending the pellets with MB, adding a little at a time (~1ml at a time) with thorough mixing to prevent the formation of lumps. Mix until drops of myosin solution are clear. Measure the volume and the [protein] (in MB) of the myosin solution
NOTE: If lumps do form while resuspending the myosin pellet, gently stir the solution overnight on a magnetic stirrer in the coldbox to dissolve the lumps. Measure the volume and the [protein] (in MB) of the myosin solution.
Volume =
D.F. =
A280 =
A320 =
A280A320
[mg/ml] = ------x =
0.56
Add MB to a final concentration of 25 mg/ml if necessary Measure the new total volume
volume MB to add = [mg/ml]/25)*volume volume =
Volume (V”) =
12.For final myosin extraction. Polymerize any remaining action by bringing the solution to .0.45M KCl, 6mMMgCl2 and 5mM ATP (pH 7.0).
0.45M KCl
.006M MgCl2
0.005M ATP
First, mix together the appropriate amounts of KCl and MgCl2 , then slowly add them to the myosin solution, mixing well. Then add the ATP and stir gently for a few minutes. Centrifuge the solution for 2 hr./100,000 x g/4C to pellet and remove the F-actin. Collect and save the supernatant containing purified myosin in a beaker. Measure the volume.
Notice: The myosin will be dissociated from the actin in the presence of ATP and will thus remain in the supernatant during the spin.
Volume=
13.Dialyze the purified myosin (20 mg/ml) vs. 4L MB in the coldbox to remove the nucleotides (ATP, ADP, P1). 3-4 changes, 4-8 hours in duration will be required.(20L MB total)
THIS IS A GOOD STOPPING POINT!!!
14.Read the [myosin] after dialysis to determine if nucleotides have been removed.
To determine if all the nucleotides have been removed by the dialysis, scan a 1mg/ml aliquot of myosin in MB from 245nm to 360 nm. The nucleotides absorb at 260nm and will mask a protein trough at 250nm, which should be about 50% of the protein peak absorbance at 280nm in the absence of nucleotide. (see graph attached).
15.If nucleotides remain present, repeat dialysis with 2-changes. Read [myosin].
16.Once the nucleotides have been removed, transfer the myosin form the dialysis tubing into a beaker on ice. Measure the final concentration (in MB) and volume of the myosin.
Final Volume (F.V.) =
D.F.=
A280=
A320=
A280A320
[mg/ml] = ------x =
0.56
17.Add an equal volume of cold glycerol to a buffered solution of myosin and mix thoroughly. Store the glycerinated myosin @20oC Label with myosin prep #, name, date and [myosin] and date rabbit was killed. Record in Myosin preps. notebook!
NOTE: Air bubbles should be removed before storing:
Notes on myosin solubility
Myosin dissolves in pure water, forming a very loose clear gel. Solubility goes through a minimum as the ionic strength is increased. The position and depth of the minimum depends on the nature of the anions, cations and the pH. (The min for KCl is near 40mM at pH 6.5-7.5.) Myosin is always handled at a pH greater than isoelectric pH (5.7 at zero salt) and thus always has a net negative charge.
Ghosh and Mihalyi (1952), Arch. Biochem. Biophys., 41, 107-117.
Brahms and Brezner (1961), Arch. Biochem. Biophys., 95, 219-228.
In solutions with strongly negative polyvalent anions (ADP, ATP, PPi, etc.) myosin stays dissolved.
Notes on myosin stability
Primary problems are irreversible aggregation and loss or changes of ATPase activity. These are associated with loss of titratable sulphydryl groups, due to oxidation. Oxidation is catalyzed by heavy metals and is accelerated by higher temp. The process is stopped by the use of ultra pure reagents and O2-free water. The process is slowed and partially reversed by chelators (to trap metals) and “sulphydryl reagents” (e.g. DTT, Beta-mercaptoethanol), low temp. and low [O2]. Microbial growth and effects of contaminating proteases are slowed or stopped by storage in glycerol at low temperatures, and by the use of NaN3 (less than 0.5mM O.K.) during normal handling.
Myosin from Myofibrils2.doc4/12/2002 10:23 AM1