Assay for hyaluronidase!!!

Jaco de Ridder j_r at scientia.up.ac.za
Fri Jul 25 09:10:38 EST 1997


Hello.

I'm a post-graduate student in Biochemistry at the University of
Pretoria, South Africa.
I'm trying to isolate an enzyme, hyaluronidase and are using the
microplate assay for this enzyme as done by Homer KA et al (Homer KA et
al, (1993), Analytical Biochemistry, 214, 435-441.). The authors of this
article made an improvement on the method of Benchetrit et al
(Benchetrit LC et al (1977), Analytical Biochemistry, 79, 431-437.).
Hyaluronic acid (HA) binds to a dye Stains-All
(1-ethyl-2-[3-(ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho-[1,2-d]thiazolium
bromide) that has a shift in spectra to 650nm. The Dye is a cationic
carbocyanine and the HA is anionic polymer.  Homer et al found that
water has to be added for this shift to take place.

What is the reason for this? Is it that something is interfering with
the dye-HA complex forming?
Problem???? The amount of water that Homer et al adds is not always
enough for the shift in spectra when I do the experiment. What is the
reason for this? Am I doing something wrong? Does temp. play a role in
the Dye-HA complex forming?

If anybody has any idea what can be done to overcome these problems or
answers to these questions, please let me know.
Thanks in advance!!

David Raats
Department of Biochemistry
University of Pretoria
South Africa
s9339981 at scientia.up.ac.za
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