Chapter 7: Microsomes
7.3 Chemical characterization - acid phosphatase
LEVEL I
Table 7.2 a list of chemical constituents found in
isolated lysosomes
MATERIALS
- 1 M Sodium acetate buffer, pH 5.7
- 0.1 M MgCl
- 0.05 M p-Nitrophenyl phosphate
- Microsome fraction from Exercise 7.1
- 150 mM KCl in 10 mM Tris-HCl Buffer, pH 7.4
- 0.5 N KOH
- Water bath at 30 ° C
- Spectrophotometer and tubes
- Bradford Protein assay
PROCEDURE
- Prepare a series of six test tubes containing 0.5 ml of each
of the following (total of 1.5 ml):
1 M Sodium acetate buffer
0.1 M MgCl
0.05 M p-Nitrophenyl phosphate
- Add 3.3 ml of distilled HO to each of the six
tubes. Mix well, and place in a 30 ° C water bath to
temperature equilibrate.
- 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.
- Add 0.2 ml of Tris-HCl buffer and 2.0 ml of KOH to tube #1
from step 1.
- 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.
- Add 2.0 ml of KOH to tube #2, mix and immediately read the
absorbance of the solution at 405 nm.
- 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.
- 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
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.
- 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.
- 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
|
|
|
|
|
3. 10
|
|
|
|
|
4. 10
|
|
|
|
|
5. 10
|
|
|
|
|
6. 10
|
|
|
|
|
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