Associates of Cape Cod, Inc.
Testing Excellence
Frequently Asked Questions
Technical Questions and Answers for Heparitinase, #100703
Is BSA added as a stabilizer to this product? Can heparitinase
be purchased without the BSA? Please provide all info.
Yes, it does include BSA as a stabilizer. When the next lot is made, it
can be requested without the BSA, but we cannot assure the quality (stability
without BSA is not known). 10 Vials at least would have to be ordered
to get it without BSA. There is not data available on how long the vials
would last, even with proper storage. Heparitinase regular can be stored
for up to one year at -70oC in aliquots after reconstitution.
Please provide the N-terminal amino acid sequence or the entire
sequence if available.
Not tested, no data available.
What is the recommended concentration (working concentration)
of enzyme per ml of assay? Does the researcher use the entire ul vial
to make a reaction mixture?
Dissolve the enzyme (0.1 units) in 100 ?l~200 ?1 of 0.1% BSA solution.
Use the 10 ?l of enzyme solution for the reaction. Reaction time = 5 hours.
Total reaction volume 100~200 ?l. It's recommended that total reaction
volume should be smaller for better results.
Is there protease contamination in heparitinase? If it cleaves
proteoglycans and other proteins then it is not good for his research.
Protease cannot be entirely removed from these enzymes. One has to use
protease inhibitors in the reaction mixture if protease is a problem.
Can you tell us what kind of BSA is being used with this enzyme
for staining? This researcher says his BSA is making bands that are dominating
over the heparitinase bands.
BSA fraction V from US origin. Due to stability of enzyme, less than 1
mg/vial of BSA fraction 5 is added in 0.1 unit of enzyme, which weighs
1/15 mg. This means close to 15 times more BSA than enzyme. Otherwise,
enzyme may not be stored for certain periods of time.
What is the assay for enzyme activity that can be read in visible
range?
There are some methods detecting reduced end after enzyme reaction. SKK
engineering division uses 3.5 Dinitrosalicyclic acid after enzyme reaction,
boil for 5 minutes then OD, at 545 mm. But this is very poor in sensitivity
and is not suitable for quantitative assay. They only use it as qualitative
method to see if enzyme exists or not. Scientists may know other procedures
detecting reducing end of the reactant.
What is the Molecular weight for Heparitinase?
64, 000
What is the best storage conditions for length of time for this
enzyme?
Keep at -70oC for longer storage. The stability is decided by the protein
concentration. Small amount of protein is less stable than concentrated
protein. And also we consider that some enzyme sticks to the tube walls.
Do you know the activity of the enzyme in different salt concentrations?
Researcher would like to use it in 1.5 M Na Chloride, how might this affect
the activity?
Please see the following chart; heparitinase activity may be negligible
in that high of a salt concentration.
Activity of Heparitinase Enzyme in NaCl Solution
| Na Chloride Concentration (M) | Activity % |
| 0.00 | M 100 |
| 0.50 | M 38 |
| 1.00 | M 17 |
| 1.50 | M 8 |
Can this enzyme be used in digestion protocols with Tris acetate
buffer instead of Na Acetate? Will it poison or even work?
Heparitinase has 80% activity in 50 mM Tris-Acetate buffer at pH 7.0 +
5mM Ca-Acetate, compared with sodium acetate buffer of pH 7.0 I think
you can use Tris-acetate buffer if you don't care about going down in
enzyme activity 20%.
Researcher cannot use the recommended Tris Buffer due to the
cells. They want to know if they can use these enzymes in PBS or RPMI
1640 buffers, and if so, will there be any loss of activity?
It should be no problem to use these other buffers as long as you keep
the pH around 8.0. No loss of activity is expected if pH is maintained
in this level.
Do you have a protocol or reference for the researcher to show
him HOW to use Heparan Sulfate (#400700) as a positive control for digestion?
You can dissolve 1 mg of HS (400700) in 100 ml of 100mM sodium acetate
buffer pH 7.0 containing 10mM calcium acetate and add 10mU of Heparitinase
which is dissolved in the same buffer (100?l/vial). You should incubate
the mix solution at 37oC for 10 hours.
Apart from the protocol on the product data sheet for this product,
do you have a protocol for protein digestion including recommended pH
and temperature ranges? Also, is the use of calcium solution to dissolve
the enzyme in ok (instead of BSA solution of water)?
To separate heparan sulfate from Glycoprotein or Proteoglycan with heparitinase,
use the following method
- Prepare solution (1) by dissolving l00 ml of sample in l00 ml
of 100 mM sodium acetate buffer pH 7.0 containing 10mM calcium acetate.
- Prepare solution (2) by dissolving 100 units of Heparitinase in 100m1 of l00 mM sodium acetate buffer pH 7.0 containing l0mM calcium acetate.
REACTION: Combine solution (1) with 1 ml of solution (2) and incubate at 37oC for 1-3 hours. Remember that this product has a little protease contamination, so we recommend using a protease inhibitor in your reaction.
Is heparitinase protease free?
No, it contains l-20 ng per vial of Trypsin. See chart below:
| Heparitinase Lot No | ng/vial as Trypsin |
| E89901 | 1.5 |
| E91801 | 1.5 |
| E91Z02 | 11.0 |
| E92X02 | 18.5 |
| E93501 | 7.5 |
| E94Y02 | 19.0 |
If Heparitinase is reconstituted, lyophilized, will it remain
stable for more than 1 week? If yes, what should it be reconstituted
in?
You should use 0.1% BSA Solution to dissolve. After dissolving, you
should store below -20oC. It is better not to divide into aliquots.
Storage time is now being examined below -20oC. Freezing and thawing
twice; it has 80% of activity at least for one month.
Customer wants to get the heparan sulfate of protein and would
like to know what to use to solubilize the heparitinase in?
The enzyme is lyophilized from buffer solution so you can dissolve it
in water.
Any information on enzyme activity in different levels of detergent?
| Detergent Remaining | Activity |
| Control (no detergent) | 100% |
| 0.1% Triton X-100 | 52% |
| 0.1% Tween 20 | 108% |
| 0.1% Tween 40 | 112% |
(over 100% such as 108 or 112% is not mean of some activation)
Please provide information on the use of a protease inhibitor
for heparitinase.
To protect the core protein in the process of enzyme treatment, the
following solution is recommended:
0.01 M EDTA
0.01 M Ethyl maleimide
5 mM PMSF (phenylmethanesulfonylflouride)
0.36M Pepstatin
Reference: Biochem. J., vol 191, pg 203, 1980.
How do you calculate the units per vial? How do you calculate
how much to use per experiment?
To get the units per vial, multiply the specific activity by the protein
(ex. 26.7 x .0075= 0.20). For the enzyme reaction condition: at 43oC,
0.1 unit of this enzyme will digest 5 mg of heparan sulfate completely
after 17 hours.
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