Myoglobin from Muscle

Myoglobin and hemoglobin

are vital to oxygen transport and storage in vertebrates

red

The former is present in large concentrations in muscles and is responsible for the _ color of the tissue.

Myoglobin

_ can be isolated by using ammonium sulfate precipitation on the buffered muscle extract.

Minced beef (or steak)

Cheesecloth

Centrifuge

Centrifuge tubes

Isolation and Characterization of Proteins

(NH₄)₂SO₄ crystals

70% buffer-diluted (NH₄)₂SO₄ solution, pH 7.5

REAGENTS AND MATERIALS 7

Myoglobin from Muscle PROCEDURE 1

Place 6.0 g of the minced beef (or steak) and 6 mL of the 70% (NH₄)₂SO₄ solution in a small beaker.

Myoglobin from Muscle PROCEDURE 2

Gently stir the mixture for 1 minute to release the myoglobin.

Myoglobin from Muscle PROCEDURE 3

Express the dark-red extract into a new beaker using the cheesecloth.

Myoglobin from Muscle PROCEDURE 4

Centrifuge the extract at 13,000 × g for 5 minutes.

Myoglobin from Muscle PROCEDURE 5

Transfer 1.5 mL of the supernatant into another empty centrifuge tube.

Myoglobin from Muscle PROCEDURE 6

Add ~0.3–0.35 g of the (NH₄)₂SO₄ crystals, ground to a fine powder. Mix gently until the solid dissolves. Avoid frothing.

Myoglobin from Muscle PROCEDURE 7

Centrifuge the sample again for at least 5 minutes.

Myoglobin from Muscle PROCEDURE 8

Decant off the supernatant. Describe the appearance of the isolated myoglobin residue.

Red

Myoglobin is responsible for the ____ color of muscle tissue.

Ammonium sulfate precipitation

Myoglobin is isolated from minced beef using:

Remove insoluble debris

What is the purpose of the first centrifugation step?

Supernatant

After centrifugation, the ____ is used for further precipitation.

(NH₄)₂SO₄ crystals

Which reagent is added in solid, ground form to further precipitate myoglobin?

Minced beef or steak

What meat source is used for myoglobin isolation?

Ammonium sulfate solution

What solution is mixed with minced beef to release myoglobin?

Frothing

What should be avoided while dissolving ammonium sulfate crystals?

13,000 × g

At what centrifuge force is the extract spun (in × g)?

Mix beef + solution → Strain → Spin → add Salt → Spin again → Save residue

Mix–Strain–Spin–Salt–Spin–Save