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Calculation Results of P1with Different Percentagesof Trifluoroethanol TFE indicated the percentage of trifluoroethanoladdition(V/V), C and C, indicated lysozyme concentration calculated from circular dichroism spectra and measured by weighmg, respectively

Determination of protein concentration from CD spectrum

ANALYTICAL LETTERS, no. 11 (2006): 1825-1835

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Abstract

Protein concentration can be determined from its circular dichroism(CD) spectrum and from programs that calculate protein secondary structure content from CD. The method established was successfully applied to lysozyme and a de novo designed peptide with a standard error of about 5%, which is comparable to concentrations determined from a...More

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Introduction
  • Estimatingprotein concentrationis a common task in protein chemistry. In the case of quantitative studies, an accurate determination of protein concentration

    Copyright 0 1998 by Marcel Dekker, Inc. www.dekker.com

    CAO, LAI, AND TANG is required.
  • In the case of quantitative studies, an accurate determination of protein concentration.
  • Protein concentration can be determined by UV absorbance, the Bradford or the Lowry assay, etc.
  • It is widely accepted that the accuracy of protein concentration is essential to this kind of calculations2'.
  • In our research2', the authors found that some of these programs can be applied to proteins with unknown concentrations, and at the same time, the concentration of the proteins can be deduced
Highlights
  • Estimatingprotein concentrationis a common task in protein chemistry
  • Since each protein secondary structure type has its own characteristic circular dichroism (CD) spectrum, CD has been widely used in protein conformational studies and programs have been developed to calculate secondary structuresof proteinsfrom their CD spectra[2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19].The principle ofthose programs is that the CD spectrum is a linear combination of spectra of individual secondary structures
  • Lysozyme concentration of this solution measured by UV absorbance was 0.163mg/ml.We can see the temperature influence on CD spectra
  • Calculation Results of P1with Different Percentagesof Trifluoroethanol TFE indicated the percentage of trifluoroethanoladdition(V/V), C and C, indicated lysozyme concentration calculated from CD spectra and measured by weighmg, respectively
  • We have proposed a procedure to calculate protein and peptide concentrations from their CD spectra
  • Since the CD spectrometer is very sensitive, and cells of different pathlength are available, calculatingprotein concentration from the CD spectrumcan be widely applied, especiallyfor newly separatedand de novo designedproteins and peptides whose extinctioncoefficientsare unknown, and for those sampleswhose solubility is so poor that UV spectrophotometrycould not be used
Methods
  • Procedure of Ev

    Protein- C CD SDectrum.

    (l), Choose a program for calculating protein secondary structures from CD spectrum.

    PROTEIN CONCENTRATION (2), Record CD spectra of protein or peptide in the range required by the program. (3), Give an initial value C, as protein concentration and change the unit of CD spectra as required by the program.

    (4), Use the program to calculate protein secondary structures. (5), Use C,=C,xTOT (TOT is the value of total amount of all secondary structures calculated) as protein concentration and repeat (3) and (4).

    (6),Repeat above processesuntil the total amount of all secondary structures equals one, the concentration used in that circle is the calculated concentration for the protein measured.

    Here the authors choose VARSLC(variab1e selection method) program'.
  • (l), Choose a program for calculating protein secondary structures from CD spectrum.
  • (5), Use C,=C,xTOT (TOT is the value of total amount of all secondary structures calculated) as protein concentration and repeat (3) and (4).
  • The authors choose VARSLC(variab1e selection method) program'
  • This program contains a basis set CD spectra of 33 proteins; the idea is to remove proteins from the basis set whose CD reflects parameters not found in the CD spectrum of the protein being analyzed.
  • Removing three proteins fiom the basis set at a time, the calculated results of all combinationswith three proteins removed was checked for a satisfactory solution; one or two proteins which are responsible for causing a problem are eliminated from the basis set, and the procedure is repeated until satisfactory analyses are obtained
Results
  • Table 2 shows that the calculatedamount of secondary structure components(H, A, P,T,0)and the root mean square errors(RMSE) were directly proportional to the total of calculated values of all secondary structures and were reversely proportional to the initial concentrations.
  • P1with Different Percentagesof Trifluoroethanol TFE indicated the percentage of trifluoroethanoladdition(V/V), C and C, indicated lysozyme concentration calculated from CD spectra and measured by weighmg, respectively.
  • The only program that was considered to be able to estimate protein secondary structure when its concentration was not known precisely is MLR[20].But, theoretically, the answers to the above two questions are yes
Conclusion
  • The authors have proposed a procedure to calculate protein and peptide concentrations from their CD spectra.
  • The results show that whether for natural protein, for less ordered synthesized polypeptide, for peptides with addition of trifluoroethanol, and even for protein at high temperature, evaluating the concentration of protein and polypeptide from their CD spectra is possible, and relatively reliable.
  • Since the CD spectrometer is very sensitive, and cells of different pathlength are available, calculatingprotein concentration from the CD spectrumcan be widely applied, especiallyfor newly separatedand de novo designedproteins and peptides whose extinctioncoefficientsare unknown, and for those sampleswhose solubility is so poor that UV spectrophotometrycould not be used
Tables
  • Table1: Calculation Results of Lysozyme at Different Temperatures 'Temp' indicates the temperatures at whch the spectra were recorded. C,, C,, C,, and C indicated the initial concentration, and calculated concentrations in the first calculation circle, in the second circle and the calculated concentration of lysozyme. C, was lysozyme concentration measured by UV absorbance. Error = (C-C,)/CUv
Download tables as Excel
Funding
  • Tlus work was supported by the National Science Foundation of China and the State Commission of Science and Technology of China
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