My original (non-diluted) protein concentration was 6.28mg/ml. This was diluted

Question

My original (non-diluted) protein concentration was 6.28mg/ml. This was diluted to an approximate concentration of 5mg/ml with phosphate buffer.

I am unsure of how to calculate the moles of thiol groups for each sample, for example in test tube 2 we used: 0.4ml phosphate buffer, 0.4ml ovalbumin, 1ml Guanidine HCl and 4ml H2O and got an absorbance reading of 0.012 at 412nm after addition of 0.2ml of DTNB.

The molar extinction coefficient of the nitromercaptobenzoate ion was given to be 1.36x10^4 M¯¹cm¯¹.

I'm assuming we have to use Beer-Lambert law to calculate the moles of thiol groups and I'm not sure what a sensible result would be in native vs denatured ovalbumin.

Could anyone help with this?

EXPERIMENT Preparation of ovalbumin (approx. 1 hour) 1. The laboratory staff will separate the white of an egg and you will be given a sample of at the start of the the into a plastic 50 mi tube egg provided and note the volume of the egg white (v ll). Place the tube on ice. Stir the white gently with a glass rod to break up the membranes. While stirring continuously add slowly an equal volume of saturated ammonium sulphate. Centrifuge (10 minutes at 4000 r.p.m. on a bench-top centrifuge) then remove and KEEP the supernatant by using a pasteur pipette to transfer the supernatant solution into a clean tube. Discard the precipitate To the supernatant solution. add o H2so4 dropwise the solution turns cloudy Land the precipitate forms. At that point the pH of the solution should be at around pH 4.6. You will be provided with pH measuring strips, but if you wish to check your pH by f you drop the pH too far, the using pH meter do ask a demonstrator for help precipitate may redissolve! centrifuge (as above) and carefully decant (pour out) and scard the supernatant but keep the precipitate To the precipitate, add 10ml of dHzo. Vortex vigorously to dissolve the Measure the absorbance at 280nm in a UV cuvette. If is off-scale, take 1 the reading ml of your ovalbumin solution and prepare a 1:10 dilution in dH20 and try again calculate the protein concentration of your original (non-diluted) sample by using the relationship 1mg.ml 1 protein has an A280 of 1.0 a Dilute your partially purified ovalbumin preparation to an approximate concentration of 5mg.ml 1 with the phosphate buffer provided 7.2). This solution will be used for part 2 below 2. Determination of thiol groups hour) Set up a series of test tubes according to the details outlined in the Table below: Test Tube No 1 (volumes in 0.4 0.4 0.4 Phosphate buffer Ovalbumin Guanidine. HCI (6.5M) H2O absor banc values

Explanation / Answer

Dear Student,

Ellman's reagent is usedfor the modification of free thiols in proteins. It rapidly forms a disulfide bond with the thiol and releases a thiolate ion which is colored. The maximal absorbance of this thiolate is at 412 nm. It's presence can be plotted against a standard curve to determine the total amount of free thiols in proteins.

Determine the molar concentration of thiols based on the molar extinction of reduced Ellman's reagent (13,600). In other words, divide the absorbance at 412 nm by 13,600. This is the molar thiol concentration. Then divide by the molar concentration of the chitosan. This will give number of thiols per chitosan molecule. Or you could use cysteine standards treated the same way as your samples and read the thiol concentration off the standard curve. If the thiols contribute significantly to the molecular weight of the conjugate, you will have to compare the weight of the conjugate to an appropriate control.

or,

use following Procedure:

1. 0.5 mg of conjugate hydrated in 250 uL of milli pore water (i.e., 2mg/mL concentration).

2.       Then add 250 uL of 0.5 M Phosphate buffer (pH 8.0).

3.       Then add 500 uL of Ellman’s reagent.

4.       Incubate the sample for 3 hrs at room temp. in dark.

5.       Measure the absorbance at 412 nm.

Regards

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