CIRCULAR DICHROISM SPECTROSCOPY (CD)

Circular Dichroism (CD) is a type of absorption spectroscopy that can provide information on the structures of many types of biological macromolecules

Higher Order Structure analysis by CD Spectroscopy

It measures the difference between the absorption of left and right handed circularly-polarized light by proteins. CD is used for;

  • Protein structure determination.
  • Induced structural changes, i.e. pH, heat & solvent.
  • Protein folding/unfolding.
  • Ligand binding
  • Structural aspects of nucleic acids, polysaccharides, peptides, hormones & other small molecules.

HIGHER ORDER STRUCTURE ANALYSIS

SECONDARY STRUCTURE ANALYSIS

TERTIARY STRUCTURE ANALYSIS

In the “Far-UV” spectral region (190-250 nm) peptide bonds produce CD signals that are sensitive to the overall secondary structure of the protein. By analyzing the CD spectra of Far-UV region, a defined assessment of the components of the protein secondary structure can be made. 

In the “near-UV” spectral region (250-350 nm) aromatic amino acids and disulfide bonds produce CD signals that are sensitive to the overall tertiary structure of the protein. By analyzing the CD spectra of near-UV region,
a good assessment of the tertiary structure of the protein sample can be made.  

HIGHER ORDER STRUCTURE ANALYSIS

HIGHER ORDER STRUCTURE ANALYSIS

SECONDARY STRUCTURE ANALYSIS​

 

 

-ve band (nm)

+ve band (nm)

α- helix

222,208

192

β- sheet

216

195

β- turn

220-230 weak

180-190 strong

205

Random Coil

200

 

TERTIARY STRUCTURE ANALYSIS

 

 

band (nm)

Phenyl alanine

250-270

Tyrosine

270-290

Tryptophan

280-300

Disulphide bonds

Broad signal throughout Near UV region

HIGHER ORDER STRUCTURE ANALYSIS

SECONDARY STRUCTURE ANALYSIS

FAR- UV-CD SPECTRA OF Bevacizumab

FAR- UV-CD SPECTRA OF CRM 197

Higher Order Structure analysis by CD Spectroscopy

TERTIARY STRUCTURE ANALYSIS

FAR- UV-CD SPECTRA OF Bevacizumab

FAR- UV-CD SPECTRA OF CRM 197

CD ANALYSIS AT CBA

 

 

Secondary structure analysis by Far UV-CD spectroscopy 

 

Tertiary Structure Analysis of Protein using Near-UV CD Spectroscopy 

Sample requirement

Amount- 0.3-1 ml (0.1-1 mg/ml)

Formulation buffer- 5 ml

Amount- 1-2 ml (1-2 mg/ml)

Formulation buffer- 5 ml

Deliverables

CD Spectra, Secondary structure analysis (relative amount of α-helix, β-sheet and random coil) and MRE (Θ in grad cm² dmol-1)

CD Spectra, MRE (Θ in grad cm² dmol-1)

Information required from Client

• Molecular weight 

• Number of residues 

• Concentration 

• Buffer composition (in case of formulation)

 This information is essential for the calculation of the optical constants and secondary structural analysis. 

 Note: If the buffer contains substances with high absorbance in the measurement range (e.g. Glycine, Citrate, Benzyl alcohol etc.), this may lead to an overload of the detector and limited measurement range.

SERVICES OFFERED BY CBA

  • Protein Intact Mass Analysis using HRMS
  • Peptide Mapping using HRMS
  • Post Translational Modifications Analysis using HRMS
  • Host Cell Protein Impurity Identification using HRMS
  • Host Cell Protein Impurity Profiling using SDS-PAGE
  • Secondary Structure Analysis using far-UV CD Spectroscopy
  • Tertiary Structure Analysis using near-UV CD Spectroscopy
  • Tertiary Structure Analysis using Fluorescence Spectroscopy
  • Higher Order Structure Analysis using Fluorescence Spectroscopy
  • Secondary Structure Analysis using FTIR Spectroscopy
  • Higher Order Structure Analysis using DSC
  • Aggregate/Multimers Analysis using SEC-MALS
  • Protein Protein Interaction Analysis using SPR
  • Biosimilarity assessment Project

SERVICES OFFERED BY CBA

Dr. Smita Kale

PI- Center for BioPharma Analysis

Venture Center

100, NCL Innovation Park, Dr. Homi Bhabha Road, Pune – 08

Phone: 020-2586-5877 | Mobile: +91-9923147592

Email: smita@venturecenter.co.in

Mr. Yashodhan Shende

Business Development Executive

Venture Center

100, NCL Innovation Park, Dr. Homi Bhabha Road, Pune – 08

Phone: 020-2586-5877 | Mobile: +91-7755920096 

Email: yashodhan@venturecenter.co.in

For more information, please contact:

Website: https://bioanalysis.in/