Circular dichroism spectroscopy (abbreviated as CD) is the most widely used method for determining the secondary structure of proteins. It is a fast, simple, and accurate method for studying protein conformation in dilute solutions. It can be measured in a solution state, which is closer to its physiological state. Moreover, the determination method is quick and simple, and it is sensitive to conformational changes, so it is one of the main methods for studying the secondary structure of proteins, and it has been widely used in the study of protein conformation.
Introduction
An optical spectrum used to infer the configuration and conformation of asymmetric molecules. The absorption coefficients (ε) of optically active materials for left-handed and right-handed circularly polarized light constituting plane polarized light are not equal, and εL ≠ εR, that is, they have circular dichroism. If the wavelength λ of plane polarized light of different wavelengths is used as the abscissa, and the difference in absorption coefficient Δε = εL-εR is plotted as the ordinate, the resulting spectrum is the circular dichroism spectrum, or CD for short. If a chiral compound absorbs in the ultraviolet-visible region, a characteristic circular dichroism spectrum can be obtained. Since εL ≠ εR, the transmitted light is no longer plane polarized light, but elliptically polarized light. The relationship between the molar ellipticity [θ] and Δε is: [θ] = 3300Δε. Circular dichroism chromatography can also plot the molar ellipticity as the ordinate and the wavelength as the abscissa. Since Δε has positive and negative values, circular dichroism also has positive circular dichroism with peaks and negative circular dichroism with valleys. Circular dichroism and spin spectra are measured in the ultraviolet-visible region, the purpose of which is to infer the configuration and conformation of organic compounds
Sample requirements
- The sample must maintain a certain purity without impurities that absorb light, and the solvent must not absorb interference at the measurement wavelength; the sample can be completely dissolved in the solvent to form a uniform and transparent solution.
- Control of nitrogen flow
- Buffer and solvent requirements and pool selection: buffer and solvent should be separately checked before preparing the solution to see if there is absorption interference in the measurement wavelength range, to see if precipitation and colloid are formed; in protein measurement, often Choose a phosphate system with excellent transparency as a buffer system.
4 Sample concentration and pool selection
Different samples have different circular dichroism spectrum ranges, and different requirements for the selection of the size (light path) and concentration of the pool. Protein CD spectroscopy measurements are generally performed in relatively dilute solutions.The band width is chosen to be 1 nm. For high-resolution measurements, a narrower slit width is used. At this time, the voltage of the photomultiplier tube is higher, and the signal-to-noise ratio of the spectrum is poor. Although the optimal band width for normal measurement is 1 ~ 2 nm, in the following cases, a wider slit width is required at the expense of resolution. When the absorbance of the sample is high but the CD signal is weak, on the one hand, try to ensure that the concentration required for measuring the CD peak is sufficient, and on the other hand, set a wider slit. However, special care should be taken at this time, because the sample may have some artifacts caused by fluorescence or stray light when the absorbance is too high (A> 2). In addition, a large slit width (generally> 2 nm) is also required when testing solid-state CD spectroscopy.
(2) Both the ellipticity and the molar ellipticity depend on the measurement conditions. Therefore, temperature, wavelength and sample concentration should always be specified.
(3) When the solid powder sample is tested by the tabletting method or the paraffin oil grinding method, it should be ground as much as possible to obtain fine and uniform sample particles. When using the paraffin oil paste method, it must be noted that some hydrophobic organic compounds may be soluble in the paraffin oil, and the CD spectrum obtained at this time should be regarded as a solution CD spectrum in a certain sense. When using the tablet method to test the solid CD, while ensuring that the qualitative concentration of the chiral sample reaches the detection requirements of the CD spectrometer, the thinner the film, the better the transparency (but avoid damage). In some cases, the tabletting method is not suitable for the determination of solid-induced CD spectra in the presence of chiral counter anions.
principle
Light is a transverse electromagnetic wave, a ray that vibrates in all directions. The electric field vector E and the magnetic field vector H are perpendicular to each other and perpendicular to the propagation direction of the light wave. Since it is mainly the electric field vector that produces the photosensitive effect, the electric field vector is generally used as the vibration vector of the light wave. The plane formed by the electric field vector of the light wave and the propagation direction is called the vibration surface of the light wave. If this vibration plane does not change with time, this beam of light is called plane polarized light, and its vibration plane is called polarization plane. The plane polarized light can be decomposed into two circularly polarized lights with the same amplitude and frequency and opposite rotation directions. The one in which the electric vector rotates clockwise is called right-handed circularly polarized light, and the one in the counterclockwise direction is called left-handed circularly polarized light. Polarized light with the same amplitude and frequency but opposite rotation direction can also be combined into a plane polarized light. If the amplitude (intensity) of the two beams of polarized light is not the same, the synthesized beam will be elliptically polarized.
About us
Pronalyse is a newly established division of Creative Proteomics, which is an integrated CRO company with rich experience in providing drug development service for over 10 years. The name Pronalyse is derived from “professional” and “analyze”. We are dedicated in providing professional analysis service in biopharmaceutical development, with the most advanced technology: protein ubiquitination, deamidation, sumoylation, phosphorylation, within the shortest turnaround time, and at the most competitive price.
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Recent Developments and Future Outlook 5.3.
Recent Developments and Future Outlook COMPANY PROFILES: PROTEOME PROFILING SERVICE PROVIDERS IN ASIA PACIFIC AND REST OF THE WORLD7.1.
Recent Developments and Future Outlook COMPANY COMPETITIVENESS ANALYSIS8.1.
Proteome Profiling Services Market, 2020-2030: Geographical Distribution12.3.4.
Proteome Profiling Services Market in Rest of the World, 2020-2030 IMPACT OF THE COVID-19 PANDEMIC ON PROTEOME PROFILING SERVICES MARKET13.1.
