NVoy Technology in Protein Circular Dichroism (CD)

Biophysical analysis of protein solutions provides essential information about the structure and behaviour of a
protein. Unfortunately protein instability can lead to denaturation or aggregation, which either prevents subsequent
analysis or gives misleading data. The addition of NV10 to freshly prepared samples can stabilise protein solutions
destined for circular dichroism spectroscopic analysis. NV10 has good transparency in the far-UV range and does
not affect protein secondary structure, but can prolong protein stability and inhibit aggregation. Unlike detergents,
NV10 is also compatible with analytical techniques including mass spectrometry and so samples prepared for CD
are still suitable for subsequent analysis.

PROTOCOL
Aggregation and stability can be very protein specific, but a general protocol is given below.

1. Determine the protein concentration (using eg. Bradford assay, BCA assay, absorbance at 280nm).
2. Typically a fivefold excess, by mass, of NV10 will protect the target protein. For example, use 100 μg/ml
    NV10 for 20 μg/ml protein.
3. Each Stabil-P.A.C. tube contains 1.25 mg NV10 as a lyophilised powder.
4. Add the protein solution to NV10 in Stabil-P.A.C. tubes to get the desired concentration, or make up a
    2.5 mg/ml stock of NV10 (1X stock) by adding 500 μL of buffer or distilled water to each Stabil-P.A.C.
    tube and then add this stock to the protein solution.
5. This protein / NV10 solution will be suitable for near and far-UV CD spectroscopy.
6. NV10 1X stock solution can be stored for 1 week at 4 oC or for longer term at -20 oC.

Troubleshooting
• If the protein shows signs of aggregation or heavy losses the NV10 to protein concentration ratio can be
    increased, ie increase NV10 concentration and / or reduce protein concentration.
• Alternatively, a lower NV10 to protein ratio can be used with proteins which have no history of
    aggregation.
• Always measure buffer blanks with buffer containing NV10.

EXAMPLE : Use of NV10 in CD Analysis
Citrate synthase catalyses the first step in the citric acid cycle. Samples stored in dilute solution at 4 oC gradually
lose activity. This is accompanied by a decrease in intensity in the far-UV CD spectrum. The addition of NV10 to
citrate synthase samples destined for CD analysis stabilises the protein without altering the secondary structure.

Citrate sythase was prepared at 18.6 μg/ml in either 5 mM Tris pH 7.8 alone, or in 5 mM Tris pH 7.8 containing
186 μg/ml NV10. Far-UV CD spectra of these samples were recorded at 22 oC in a 1 cm pathlength quartz cuvette.
Buffer blanks containing either 5 mM Tris pH 7.8 alone, or 5 mM Tris pH 7.8 containing 186 μg/ml NV10 were also
recorded, and the blank spectra were subtracted from the sample spectra. The samples were stored at 4 oC for
96 hours, then allowed to warm to room temperature and the far-UV CD spectra were measured again.

The initial far UV CD spectrum of citrate synthase was not affected by the presence of NV10, either by loss of
transparency at low wavelength or perturbation of secondary structure. While the samples containing citrate
synthase alone had lost signal intensity over the storage period suggesting loss of either material or secondary
structure, the citrate synthase protected by NV10 retained virtually the full intensity of the far-UV CD signal. This
observation is accompanied by reduction of the enzyme activity for citrate synthase alone, and retention of enzyme
activity for citrate synthase in the presence of NV10 after storage for 96 hours at 4 oC.

Summary
NV10 can protect protein structure and maintain stability in solution prior to CD analysis.