Chapter 7: Microsomes

7.3 Chemical characterization - acid phosphatase

LEVEL I

Table 7.2 a list of chemical constituents found in isolated lysosomes

MATERIALS

PROCEDURE

  1. Prepare a series of six test tubes containing 0.5 ml of each of the following (total of 1.5 ml):

  2. Add 3.3 ml of distilled H_2O to each of the six tubes. Mix well, and place in a 30 ° C water bath to temperature equilibrate.

  3. Prepare a serial dilution of your lysosome fraction by adding 1.0 ml of lysosome suspension from Exercise 7.1 to 9.0 ml of 150 mM KCl/10 mM Tris-HCl Buffer. Mix well and add 1.0 ml of the diluted suspension to a new tube containing 9.0 ml of Tris- HCl buffer. Mix and repeat the dilutions two more times. In addition to the undiluted lysosome suspension, label the diluted fractions as 1/10, 1/100, 1/1,000 and 1/10,000.

  4. Add 0.2 ml of Tris-HCl buffer and 2.0 ml of KOH to tube #1 from step 1.

  5. Turn on a spectrophotometer, adjust the wavelength to 405 nm and use the sample in tube #1 to blank the instrument.

    6. Add 0.2 ml of the undiluted the lysosome fraction to tube #2, mix and place in water bath at 30 ° C for exactly 5 minutes.

  6. Add 2.0 ml of KOH to tube #2, mix and immediately read the absorbance of the solution at 405 nm.

  7. The absorbance should be between 0.3 and 0.4. If not, repeat steps 6 and 7 for the diluted samples, starting with the 1/10 in tube #3 and continuing through the 1/10,000 dilution in tube #6. Each should be done separately, one at a time to prevent incorrect timing of the reaction.

  8. Using the absorbance reading for the dilution which yields an absorbance change of 0.3-0.4 in five minutes, determine the actual rate of absorbance change for that dilution.

    10. Convert the absorbance change to a rate of p-Nitrophenyl phosphate conversion to p-nitrophenol. Use the Beer-Lambert law, with an extinction coefficient = 18.8 x 10^6 Abs. Units/mole of p-nitrophenol.

    Convert all absorbance readings to micromoles of p-nitrophenol formed in five minutes. Divide by the time (5 minutes) to calculate micromoles formed per minute.

  9. Determine the concentration of protein in the dilution from step 9, using the Bradford protein determination ( Appendix G ) and bovine serum albumin as the standard.

  10. Determine the rate of enzyme activity per mg. protein present in the diluted fraction.

    Tube/Dilution
    Protein Content
    Absorbance(5')
    [p-nitrophenol]
    Activity/minute
    1. Blank
    ____
    0
    ____
    ____
    2. 10^0




    3. 10^-1




    4. 10^-2




    5. 10^-3




    6. 10^-4




OPTIONAL

Enzyme activity and protein concentration can be measured for each step in the centrifugation and isolation of lysosomes. Assuming that acid phosphatase is an effective measure of the purity of lysosomes, the increased purity during separation can be monitored by measuring the increased enzyme activity per mg of protein. Lysosome purification can be monitored to yield the maximum activity per mg. protein. While lysosomes are best characterized by the presence of acid pyrophosphorylases, they contain a large number of functional enzymes.

Table 7.2 presents a list of chemical constituents found in isolated lysosomes.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu