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Topic: NADH binding to LDH  (Read 2763 times)

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Offline sebster

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NADH binding to LDH
« on: November 09, 2014, 05:44:49 AM »
Hi everyone, Im completely stuck in interpreting this data. Any help would be greatly appreciated.

Reduced NAD emits fluorescent light at 460 nm when it is excited at 340 nm, and this fluorescence has been found to be enhanced in the presence of lactate dehydrogenase. This effect was used to determine the stoichiometry of binding.

To 2ml of a 0.473 mg/ml solution of beef-heart lactate dehydrogenase in a 0.05M Tris-acetate buffer, pH 7.2, were added small amounts of 10-3 M NADH solution in the same buffer.
The fluorescence emission at 460 nm was measured (in arbitrary units) after each addition and, as a control, readings were also made of the fluorescence changes when the NADH was added to 2ml of the buffer

http://imgur.com/qrWhDqU

The concentrations of protein in the effluent were estimated by measurements of the absorbance at 280 nm, and the volume of buffer necessary to elute each protein form the column was thus determined.

http://imgur.com/5tEaq9o
Thank you so much
« Last Edit: November 09, 2014, 07:53:30 AM by Arkcon »

Offline Arkcon

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Re: NADH binding to LDH
« Reply #1 on: November 09, 2014, 07:51:39 AM »
Hi everyone, Im completely stuck in interpreting this data. Any help would be greatly appreciated.

Well, I'm completely stuck as to the question -- you haven't asked one. ;)

Quote
Reduced NAD emits fluorescent light at 460 nm when it is excited at 340 nm, and this fluorescence has been found to be enhanced in the presence of lactate dehydrogenase. This effect was used to determine the stoichiometry of binding.

OK, this is a clear statement.  Is this the question?  You want to determine stoichiometry of binding?  Can you start by showing you know something of the topic?  Start with the formula from your textbook, and try to see what you know, and what you still need to compute from your experimental data.  This is specified in the Forum Rules{click}.

Quote
To 2ml of a 0.473 mg/ml solution of beef-heart lactate dehydrogenase in a 0.05M Tris-acetate buffer, pH 7.2, were added small amounts of 10-3 M NADH solution in the same buffer.
The fluorescence emission at 460 nm was measured (in arbitrary units) after each addition and, as a control, readings were also made of the fluorescence changes when the NADH was added to 2ml of the buffer

http://imgur.com/qrWhDqU

OK, show us you can use this tabular data.

Quote
The concentrations of protein in the effluent were estimated by measurements of the absorbance at 280 nm, and the volume of buffer necessary to elute each protein form the column was thus determined.

http://imgur.com/5tEaq9o
Thank you so much

Column?  Elution?  That wasn't in the set up of this problem so far.  Is that the question?
Hey, I'm not judging.  I just like to shoot straight.  I'm a man of science.

Offline Babcock_Hall

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Re: NADH binding to LDH
« Reply #2 on: November 10, 2014, 10:22:12 AM »
@OP, What information do you think you can find from the Sephadex G-200 data?

Offline sebster

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Re: NADH binding to LDH
« Reply #3 on: November 17, 2014, 01:57:47 PM »
Sorry everyone, the question is to simply comment on the significance of these results, and if you could suggest any further experiments to verify your conclusions?

Offline Babcock_Hall

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Re: NADH binding to LDH
« Reply #4 on: November 17, 2014, 03:58:57 PM »
It is a forum rule that you must show your attempt at doing a problem before we can help you.

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