CDNB in GST estimation

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questioner
questioner's picture
CDNB in GST estimation

Hi,
I am trying to estimate the amt of Glutathione-S-transferase in the serum of malaria-infected mice.
I used 1mM reduced glutathione+buffer+25microL CDNB(chloro-dinitro benzene)+Post-mitochondrial Supernatant(PMS). I then measured OD at 340nm.
I want to know why we add CDNB in this assay.
Thanks.
Q

Woodpecker
Woodpecker's picture
questioner wrote:Hi,

questioner wrote:

Hi,
I am trying to estimate the amt of Glutathione-S-transferase in the serum of malaria-infected mice.
I used 1mM reduced glutathione+buffer+25microL CDNB(chloro-dinitro benzene)+Post-mitochondrial Supernatant(PMS). I then measured OD at 340nm.
I want to know why we add CDNB in this assay.
Thanks.
Q

CDNB is a chromogenic substrate for GST.
see Mannervik, B. and Danielson, U.H., CRC Crit. Rev. Biochem. 23, 283 (1988).

shylet
shylet's picture
GST's are drug metabolizing

GST's are drug metabolizing enzymes which catalyze the addition of a glutathione moety to lipophilic molecules making them more water soluble. CDNB is a synthetic GST substrate. The GST assay you are referring to measures the conjugation of CDNB with reduced glutathione. This conjugation increase the wavelength to 340nm. The increase in absorbance correlates to amount of glutathione in the sample.

Thongchai
Thongchai's picture
Hi, I just a second year in

Hi, I just a second year in master degree of science, biochemistry and i dont have much skill in his field because i switch from biology in BS. So i'm interesting in Glutathione enzyme family so i mention about GST, too. I quite well to understand why to use CDNB but another seriuosly problem for is how can i calculate the unit of GST in the human blood.

All protocol or papers or Kit assay, alway propose in different way to estrablish the value of enzyme activity unit, but i can follow their equations. So please anyone can help me. !

i setup the condition that consist of 0.8 ml 0.1 M phosphate buffer, 0.1 ml 5 mM GSH and 0.1 ml 2 mM CDNB, and monitor ed it in 340 nm by Shimadzu UV 160 (spectrometer) for 5 min with 25 C. I detected in blood sample with dilution about 100 (were used for dilution factor). I setup blank with PBS instead blood. So after i monitor it, the resulted shown me like his
(kinetic = Abs/min)
: blank = 0.068
: sample = 0.081
: sample = 0.089

So, I have some question here
1. Why blank solution had high value?
2. How can i calculate the enzymeatic activity

first of all, i would like to show the some equation of my opinion

((Abs/min of sample - Abs/min of blank)/9.6)*(1/0.1)*(df) = mmol/min/ml (U)

9.6 = extinction coefficient
1 = whole volume
0.1 = sample volume
df = dilution factor = 100

after that, i used another step to make unit in U/mg Hb

so here, U/mg Hb

my second question is about Hb content
I study in hosptol so I measure Hb content via department in complete blood count unit and i got 14.4 g/dl

Can i use this value immediately for complete equation or i must be change it in mg, or i must be make it in term of volume of blood sample that i used (i used blood only 10 uL and diluted it to 1000 uL: 100X- and used in assay only 100 uL)

so, of any one hear me or read this passage, please forgive me with my worse English, and hopefully may be there are someone can understand and help me to solve the ploblem and make me know more about the technique or another notice for work through and pass through my master degree thesis.

Thank U, for any help or any read.
I'm in Thailand, and may be i cant access internet for a while so please answer me in my e-mail - eval(unescape('%64%6f%63%75%6d%65%6e%74%2e%77%72%69%74%65%28%27%3c%61%20%68%72%65%66%3d%22%6d%61%69%6c%74%6f%3a%69%6d%70%72%65%73%73%69%6f%6e%74%69%63%6b%40%79%61%68%6f%6f%2e%63%6f%6d%22%3e%69%6d%70%72%65%73%73%69%6f%6e%74%69%63%6b%40%79%61%68%6f%6f%2e%63%6f%6d%3c%2f%61%3e%27%29%3b'))

:)

frangky
frangky's picture
After read your topic, i have

After read your topic, i have two questions,
firstly,  why you added so high concentration of CDNB and GSH(2mM&5mM, or it is not the final concentration?), as the CDNB and GSH may conjugate without GSTs, so the blank solution had high value.
secondly, in your reaction system, the whole was 1 ml, 0.8ml PBS + 0.1mlCDNB + 0.1mlGSH, so where is the sample?