<< Chapter < Page Chapter >> Page >

Sample preparation and starting the instrument

Most of proteins and peptides will require using buffers in order to prevent denaturation. Caution should be shown to avoid using any optically active buffers. Clear solutions are required. CD is taken in high transparency quartz cuvettes to ensure least interference. There are cuvettes available that have path-length ranging from 0.01 cm to 1 cm. Depending on UV activity of buffers used one should choose a cuvette with path-length (distance the beam of light passes through the sample) that compensates for UV absorbance of buffer. Solutions should be prepared according to cuvette that will be used, see [link] .

Choosing the appropriate cuvette based upon the sample concentration.
Cuvette path (cm) Concentration of sample (mg/mL)
0.01 - 0.02 0.2 – 1.0
0.1 0.05 – 0.2
1 0.005 – 0.01

Besides, just like salts used to prepare pallets in FT-IR, the buffers in CD will show cutoffs at a certain point in low wavelength region, meaning that buffers start to absorb after certain wavelengh. The cutoff values for most of common buffers are known and can be found from manufacturer. Oxygen absorbs light below 200 nm. Therefore, in order to remove interference buffers should be prepared from distilled water or the water should be degassed before use. Another important point is to accurately determine concentration of sample, because concentration should be known for CD data analysis. Concentration of sample can be determined from extinction coefficients, if such are reported in literature also for protein samples quantitative amino acid analysis can be used.

Many CD instrument come bundled with a sample compartment temperature control unit. This is very handy when doing stability and unfolding/denaturation studies of proteins. Check to make sure the heat sink is filled with water. Turn the temperature control unit on and set to chosen temperature.

UV source in CD is very powerful lamp and can generates large amounts of Ozone in its chamber. Ozone significantly reduces the life of the lamp. Therefore, oxygen should be removed before turning on the main lamp (otherwise it will be converted to ozone near lamp). For this purpose nitrogen gas is constantly flushed into lamp compartment. Let Nitrogen flush at least for 15 min. before turning on the lamp.

Collecting spectra for blank, water, buffer background, and sample

  1. Collect spectrum of air blank ( [link] ). This will be essentially a line lying on x –axis of spectrum, zero absorbance.
  2. Fill the cuvette with water and take a spectrum.
  3. Water droplets left in cuvette may change concentration of your sample, especially when working with dilute samples. Hence, it is important to thoroughly dry the cuvette. After drying the cuvette, collect spectrum of buffer of exactly same concentration as used for sample ( [link] ). This is the step where buffer is confirmed to be suitable spectrum of the buffer and water should overlap within experimental error, except for low wavelength region where signal-to-noise ratio is low.
  4. Clean the cuvette as described above and fill with sample solution. Collect the CD spectrum for three times for better accuracy ( [link] ). For proteins multiple scans should overlap and not drift with time.
CD spectra of blank and water (left), buffer (center), and sample (right). Lysozyme in 10 mM sodium phosphate pH 7. Adapted by permission from N. Greenfield, Nat. Protoc. , 2006, 1 , 6.

Data handling and analysis

After saving the data for both the spectra of the sample and blank is smoothed using built-in commands of controller software. The smoothed baseline is subtracted from the smoothed spectrum of the sample. The next step is to use software bundles which have algorithms for estimating secondary structure of proteins. Input the data into the software package of choice and process it. The output from algorithms will be the percentage of a particular secondary structure conformation in sample. The data shown in [link] lists commonly used methods and comparers them for several proteins. The estimated secondary structure is compared to X-ray data, and one can see that it is best to use several methods for best accuracy.

Comparison of secondary structure estimation methods. Adapted by permission from N. Greenfield, Nat. Protoc. , 2006, 1 , 6.

Conclusion

What advantages CD has over other analysis methods? CD spectroscopy is an excellent, rapid method for assessing the secondary structure of proteins and performing studies of dynamic systems like folding and binding of proteins. It worth noting that CD does not provide information about the position of those subunits with specific conformation. However, CD outrivals other techniques in rapid assessing of the structure of unknown protein samples and in monitoring structural changes of known proteins caused by ligation and complex formation, temperature change, mutations, denaturants. CD is also widely used to juxtapose fused proteins with wild type counterparts, because CD spectra can tell whether the fused protein retained the structure of wild type or underwent changes.

Bibliography

  • L. Que, Physical Methods in Bioinorganic Chemistry – Spectroscopy and Magnetism , University Science Books, Sausalito (2000).
  • J. P, Wang, J. Nian, L. Xiaoqi, X. Yunhao, and C. S. Hanke, New J. Phys. , 2010, 12 , 063032.
  • N. Greenfield, Nat. Protoc. , 2006, 1 , 6.
  • G. Holzwarth and P. Doty, J. Am. Chem. Soc. , 1965, 87 , 218.
  • N. Greenfield and G.D. Fasman, Biochemistry , 1969, 8 , 4108.
  • P. Atkins and J. de Paula, Elements of Physical Chemistry , 4th ed, Oxford University Press (2005).
  • M. Rutherfurd and M. Dunn, Curr. Protoc. Protein Sci. , 2011, 63 , 3.2.1.

Questions & Answers

what does nano mean?
Anassong Reply
nano basically means 10^(-9). nanometer is a unit to measure length.
Bharti
do you think it's worthwhile in the long term to study the effects and possibilities of nanotechnology on viral treatment?
Damian Reply
absolutely yes
Daniel
how to know photocatalytic properties of tio2 nanoparticles...what to do now
Akash Reply
it is a goid question and i want to know the answer as well
Maciej
characteristics of micro business
Abigail
for teaching engĺish at school how nano technology help us
Anassong
Do somebody tell me a best nano engineering book for beginners?
s. Reply
what is fullerene does it is used to make bukky balls
Devang Reply
are you nano engineer ?
s.
fullerene is a bucky ball aka Carbon 60 molecule. It was name by the architect Fuller. He design the geodesic dome. it resembles a soccer ball.
Tarell
what is the actual application of fullerenes nowadays?
Damian
That is a great question Damian. best way to answer that question is to Google it. there are hundreds of applications for buck minister fullerenes, from medical to aerospace. you can also find plenty of research papers that will give you great detail on the potential applications of fullerenes.
Tarell
what is the Synthesis, properties,and applications of carbon nano chemistry
Abhijith Reply
Mostly, they use nano carbon for electronics and for materials to be strengthened.
Virgil
is Bucky paper clear?
CYNTHIA
so some one know about replacing silicon atom with phosphorous in semiconductors device?
s. Reply
Yeah, it is a pain to say the least. You basically have to heat the substarte up to around 1000 degrees celcius then pass phosphene gas over top of it, which is explosive and toxic by the way, under very low pressure.
Harper
Do you know which machine is used to that process?
s.
how to fabricate graphene ink ?
SUYASH Reply
for screen printed electrodes ?
SUYASH
What is lattice structure?
s. Reply
of graphene you mean?
Ebrahim
or in general
Ebrahim
in general
s.
Graphene has a hexagonal structure
tahir
On having this app for quite a bit time, Haven't realised there's a chat room in it.
Cied
what is biological synthesis of nanoparticles
Sanket Reply
what's the easiest and fastest way to the synthesize AgNP?
Damian Reply
China
Cied
types of nano material
abeetha Reply
I start with an easy one. carbon nanotubes woven into a long filament like a string
Porter
many many of nanotubes
Porter
what is the k.e before it land
Yasmin
what is the function of carbon nanotubes?
Cesar
I'm interested in nanotube
Uday
what is nanomaterials​ and their applications of sensors.
Ramkumar Reply
what is nano technology
Sravani Reply
what is system testing?
AMJAD
preparation of nanomaterial
Victor Reply
how did you get the value of 2000N.What calculations are needed to arrive at it
Smarajit Reply
Privacy Information Security Software Version 1.1a
Good
Berger describes sociologists as concerned with
Mueller Reply
advantages of NAA
Sai Reply
how I can reaction of mercury?
Sham Reply

Get the best Physical methods in ch... course in your pocket!





Source:  OpenStax, Physical methods in chemistry and nano science. OpenStax CNX. May 05, 2015 Download for free at http://legacy.cnx.org/content/col10699/1.21
Google Play and the Google Play logo are trademarks of Google Inc.

Notification Switch

Would you like to follow the 'Physical methods in chemistry and nano science' conversation and receive update notifications?

Ask