[rashmi]

I would like to know how high salinity (>3% NaCl) would affect enzyme substrate interactions (in vitro)

[Arkcon]

It will likely depend on the particular enzyme but you may be able to figure this one out for yourself.  (And be better off in the future if you do.)  Ask yourself:  What is an enzyme?  What is it made of?  How do they work?  What holds them together?  (If you'll let me spoil some of the answer for you.)

[faces]

What is an enzyme?

ELISA

[rashmi]

It will likely depend on the particular enzyme but you may be able to figure this one out for yourself.  (And be better off in the future if you do.)  Ask yourself:  What is an enzyme?  What is it made of?  How do they work?  What holds them together?  (If you'll let me spoil some of the answer for you.)

Lets see - an enzyme is a biocatalyst and is a protein by nature and I believe the enzyme substrate interactions depend on the physcial shape of both. So its possible that high salinity can denature the enzyme and bring down the rate of reaction.

Now to the reason why I wanted to know this. There is a product called colilert from IDEXX that we use to enumerate coliforms in water samples. We have observed that high salinity samples (Sea water & ground water) tend to give us much higher results than traditional method.

The test is an enzyme substrate reaction (APHA 9223) so I believe in high salinity waters (in our region it is usually about 5%) the enzyme gets damaged or bacterial growth is relatevly slow and not sufficient enzyme is produced. So the count should be low, but the results we get is high.

The enzymes involved are b-galactosidase & b-glucuronidase---> react with substrate (b-D-Galactopyranoside & b-D-glucuronide) and the chromophore (o-nitrophenol & 4-methyl umbelliferyl) is released give colour or fluoroscence.

Now if high salinity was making the substrate-chromophore unstable - well this can yeild high results. I am just not sure what is happening.

[Arkcon]

It would not be easy to predict.  But you can certainly check the package insert for the information provided on salinity ranges -- it should say under what range the kit can be used.  Failing that, you could ask the manufacturer, they should be able to tell you what they know, or at least, what they don't know -- as in, outside this range of salinity, our test has no been evaluated, we make no guarantees, etc.  You can always perform the experiment yourself --prepare a coliform culture, test it with this kit, then spike it with a measured amount of NaCl, and retest to see the effect. 

[rashmi]

Thanks!

We had already discussed with the manufacturer. They didn't give a reason but said that for marine waters the results were not suitable, unless diluted at least 10 times. We are currently testing with a 20 times dilution as sometimes even 10 times dilution gives a very high count.

[Pradeep]

ELISA is a techniques which enzymes are used.

Enzyme Linked Immuno Sorbent Assay