Back to the Top
Dear all,
I will do the incubation of Carbonyl reductase of my drug.
I found that some paper used the NADH, and others used NADPH.
What is the difference between them?
And both of them, has the commercial products. Somebody directly used
the NADPH or NADH, somebody used regenerating system?
What is the difference between them? Which one is better?
How to prepare the NADH-regenerating system?
Thanks,
Xiaoming
Back to the Top
See a good biochemistry text
Back to the Top
I just step into this area.
I read some of the biochemistry books.
But I find after I used the NADP and NADPH (or regeneration system), I
got confilict results from the book and references.
So this results make me confused.I need sombody share some experiences
with me. Maybe, there are some condition which I can not control cause
the problem.
Hope I can solve this problem.
Thanks,
Xiaoming
Back to the Top
The following message was posted to: PharmPK
Xiaoming,
As long as your coenzyme is in excess, you will reduce the order of the
reaction negating the effects of carbonyl reductase specificity for
NADPH
over NADH.
Regeneration requires glucose dehydrogenase for NADPH and formate
dehydrogenase for NADH.
Cheers
-Shawn
Back to the Top
Dear Shawn,
Thank you very much.
My NADH-regeneration system is composed of:
NAD+, Lactate, D-lactate Dehydrogenase, MgCl2.
The concentration of all compounds is similar with NADPH-regeneration
system
Some book said that maybe the coenzyme need the other cofactors, such
Zn2+.
Do you think I need to change the MgCl2 to ZnCl2 ?
Thanks,
Xiaoming
Back to the Top
The following message was posted to: PharmPK
Dear Xiaoming,
Depending on your source of enzyme, this could play a role in its
preference
of cofactor, but that does not mean the preference will be noticeable.
You can control for a few variables such as transition metal versus
divalent
cation in varying amounts to see if the regeneration system is
creating a
limiting condition.
But if you feel uneasy that your results are in conflict with a
literature
source, that in and of itself, is no reason modifying a protocol if it
achieves your objectives.
Even if you have identical assay conditions, transition metal cofactors,
source of enzymes, methods of data analysis, etc., you may not achieve
the
same result.
Simply keep a very detailed account of what you have done, and the
results
offer a contribution based on your chosen materials and methods.
Best of luck,
-Shawn
Back to the Top
The following message was posted to: PharmPK
Dear Xiaoming,
I mentioned the protocol of preparation of NADPH regeneration
system.
Try with this recipe you may get good results
Preparation of Solution - I:
S.No. Component Conc. For 10mL
1. Glucose-6-Phosphate 20mg/1mL 200mg
2. NADP 20mg/1mL 200mg
3. MgCl2(1M Solution) 140uL/1mL 1400uL
Note: While weighing the chemicals like NADP and Glucose - 6 - Phosphate
make sure that lights are switched off.
* Add all the above ingredients in foil wrapped 50ml conical tube and
made the volume to 10mL with autoclaved Milli - Q water.
* Make the 1mL aliquot in properly labeled aluminum foil wrapped 1.5mL
micro centrifuge tubes kept on ice. Store the aliquots at -20oC in
labeled
box.
Preparation of Sodium citrate buffer:
S.No Component Conc. For 100mL
1. Sodium citrate tribasic 100mM 2.94gms
* Weigh 2.94gms of Sodium citrate tribasic and add 90mL of a/c Milli -
Q water, allow it to completely dissolve by keeping it on magnetic
stirrer
and make up the final volume to 100mL.
* Store this buffer at RT (25oC).
Preparation of Solution - II:
5mL
S.No Component Stock For 5mL
1, Sodium citrate tribasic 100mM 250uL
2, Glucose - 6 - Phosphate Dehydrogenase 1000U/mL 200uL
* Add above reagents mentioned in the table, in foil wrapped 50mL
Conical tube and make the volume to 5mL with autoclaved Milli - Q water.
* Make 1mL aliquots in properly labeled aluminum foil wrapped 1.5mL
micro centrifuge tubes, kept on ice. Store the aliquots at -20oC in
labeled
box.
Preparation of NADPH Regeneration System, 12ul of final volume (12ul is
required. / 200ul reaction/well)
* NADPH regeneration system is the combination of Solution - I and
Solution - II.
* To be prepared at the time of performing the fluorescence assay.
* Prepare cocktail for two extra reactions than required, to take care
of pipetting error.
For each 200ul of final reaction, 10uL of Solution - I and 2 uL of
Solution
-II are required.
Thanks and Regards,
K.N.Purna chandra rao
Research Associate
PREMAS Biotech Pvt. Ltd.
Plot No. 77, Sector 4,
IMT Manesar.
Gurgaon-122050Haryana,
Want to post a follow-up message on this topic?
If this link does not work with your browser send a follow-up message to PharmPK@boomer.org with "Difference between the NADP and NADPH" as the subject | Support PharmPK by using the |
Copyright 1995-2011 David W. A. Bourne (david@boomer.org)