Infrared Protein Analysis
In infrared spectroscopy molecular vibrations are measured due to the specific absorption of infrared radiation by chemical bonds. The shape and frequency of the amide I band, which is assigned to the C=O stretching vibration within the peptide bonds of the protein molecule, is very characteristic for the structure of the studied protein. From this single band the presence and relative percentage of secondary structure elements (alpha-helix, beta-sheet) in a protein can be determined and conformational changes are detected with high sensitivity. Any alterations in the structure regardless if they were induced by temperature or pH changes, ligand binding, mutations etc. are reflected by specific absorptive changes in the amide I region.
Since IR obeys the rules of Lambert-Beer´s law, the protein spectrum can be utilized for concentration analysis. Within seconds and without any bio- or immuno-chemical staining, protein concentrations in an extremely wide range from about 0.1 mg/ml to >100 mg/ml can be determined directly from the undiluted solution.
However, the information content of IR protein spectrum is not limited to the amide I band. Side chains of various amino acids can also be monitored and directly compared under varying environmental conditions (pH, salts, ligands), or in mutant proteins with altered primary sequence.
The advantages of infrared protein analysis are its ease of use and its relatively inexpensive capability of studying proteins in aqueous media at any buffer conditions. Therefore, the IR-technique is a powerful analysis method in the pharmaceutical industry, e.g. for formulation optimization, stability studies during drug development and QC of protein drug products.
In research isolate structural effects of a protein while binding to drugs or interacting with substrates and inhibitors are followed. Furthermore, the formation of multimeric structures like aggregates or fibrils can be monitored by infrared spectroscopy (IR), even in real time.
It should be stressed that these investigations are not restricted to water-soluble proteins, but can also be performed on membrane bound proteins.
The CONFOCHECK is a dedicated Fourier transform infrared (FT-IR) spectrometer system that was developed to fulfill the demanding requirements for protein analysis in aqueous solution. Its specific concept facilitates a fast data acquisition with a high sample throughput controlled by a user friendly software interface.
Applications for CONFOCHECK:
- Protein dynamics (temperature induced conformational changes)
- Protein quantification (from 0.03 mg/ml up to > 100mg/ml in aqueous solution)
- Determination of secondary structure
- Monitoring of conformational changes during protein aggregation, precipitation and crystallization
- Quantification of all kind of solutes in aqueous samples