Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy

Henryk Mach1, C. Russell Middaugh1, Nancy Denslow2

1 Merck Research Laboratories, West Point, 2 University of Florida, Gainesville
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.2
DOI:  10.1002/0471140864.ps0702s01
Online Posting Date:  May, 2001
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Abstract

Because each protein (gene product) has a unique amino acid sequence, the particular aromatic amino acid content of each protein results in a unique spectrum in the near‐UV (250 to 350 nm) region. The highly specific microenvironment experienced by each aromatic residue in the three‐dimensional protein matrix results in fine shifts in a protein's spectrum. This unit provides protocols for the detection and analysis of UV spectra of recombinant proteins and their peptide fragments. The unique UV spectral properties of proteins can in turn be used to assess their purity. This application is inherent in the use of a diode array detector to monitor the effluent from a high‐performance liquid chromatography (HPLC) column. A protocol using this technique to assess the purity of recombinant proteins is presented.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of Proteins Using Near‐UV Spectrophotometry
  • Support Protocol 1: Interpreting Protein Near‐UV Spectra
  • Basic Protocol 2: Analysis of Proteins Using an HPLC with a Diode‐Array Detector
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Proteins Using Near‐UV Spectrophotometry

  Materials
  • Detergent solution
  • Methanol
  • 0.1 N hydrochloric acid
  • recipeAcid/ethanol cleaning solution (see recipe)
  • 10 N NaOH
  • recipe4% holmium oxide (aqueous solution in 10% perchloric acid ; see recipe)
  • Buffer identical to that containing the protein (reference standard)
  • Sample for analysis in solution at an appropriate concentration (e.g., 0.05 to 1 mg/ml; see below)
  • Vacuum‐powered cuvette washer (NSG Precision Cells, Kontes Glass, or equivalent)
  • Two factory‐matched synthetic quartz (Suprasil or equivalent) cuvettes (Hellma, NSG Precision Cells, or equivalent; for double‐beam spectrophotometers) or a single cuvette (for single‐beam use)
  • Gel‐loading plastic pipet tips (Marsh Biomedical Products; Rainin Instruments) for a pipettor (Pipetman, Eppendorf, or equivalent)
CAUTION: Employ appropriate safety precautions when using concentrated acids and bases.

Support Protocol 1: Interpreting Protein Near‐UV Spectra

  Materials
  • Buffer A: 0.1% (v/v) trifluoroacetic acid (TFA; reagent grade) in water (Milli‐Q or equivalent purity)
  • Buffer B: 0.09% (v/v) TFA in 80% acetonitrile (HPLC grade)
  • C 4 reversed phase column and guard: (e.g., Vydac C 4, 250 × 4.6‐mm‐i.d. column, 5‐µm particle size, 300‐Å pore size for samples in the 0.5 to 5 nmol range or a 250 × 2.1‐mm‐i.d. column for samples in the 100 to 500 pmol range)
  • HPLC with a diode‐array detector (e.g., Hewlett‐Packard, Waters, Beckman)
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Figures

Videos

Literature Cited

Literature Cited
   Glasel, J.A. 1995. Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratios. BioTechniques. 18:62‐63.
   Ichikawa, T. and Terada, H. 1977. Second derivative spectrophotometry as an effective tool for examining phenylalanine residues in proteins. Biochim. Biophys. Acta. 494:267‐270.
   Levine, R.L. and Federici, M.M. 1982. Quantitation of aromatic residues in proteins: Model compounds for second‐derivative spectroscopy. Biochemistry 21:2600‐2606.
   Mach, H. and Middaugh, C.R. 1994. Simultaneous monitoring of the environment of tryptophan,tyrosine and phenylalanine residues in proteins by near‐UV second derivative spectroscopy. Anal. Biochem. 222:323‐331.
   Mach, H., Thomson, J.A., and Middaugh, C.R. 1989. Quantitative analysis of protein mixtures by second derivative absorption spectroscopy. Anal. Biochem.. 181:79‐85.
   Mach, H., Middaugh, C.R., and Lewis, R.V. 1992a. Detection of proteins and phenol in DNA samples with second derivative absorption spectroscopy. Anal. Biochem. 200:20‐26.
   Mach, H., Middaugh, C.R., and Lewis, R.V. 1992b. Statistical determination of the average values of the extinction coeficients of tryptophan and tyrosine in native proteins. Anal. Biochem. 200:74‐80.
   Mach, H., Burke, C.J., Sanyal, G., Tsai, P.‐K., Volkin, D.B., and Middaugh, C.R. 1995. Origin of the photosensitivity of a monoclonal immunoglobulin G. In Formulation and Delivery of Proteins and Peptides (J.L. Cleland and R. Langer, eds.) pp. 72‐84. American Chemical Society, Washington, D.C.
   Manchester, K.L. 1995. Value of A260/A280 ratios of measurement of purity of nucleic acids. BioTechniques 19:208‐210.
   Mant, C.T. and Hodges, R.S. 1991. Requirement for peptide standards to monitor column performance and the effect of column dimensions, organic modifiers, and temperature in reversed‐phase chromatography. In High‐Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis, and Conformation (C.T. Mant and R.S. Hodges, eds.) pp. 289‐295. CRC Press, Boca Raton, Fla.
   Middaugh, C.R. and Volkin, D.B. 1992. Protein solubility. In Stability of Protein Pharmaceuticals, Part A: Chemical and Physical Pathways of Protein Degradation (T.J. Ahern and M.C. Manning, eds.) pp. 109‐134. Plenum Press, New York.
   Nozaki, Y. 1990. Determination of tryptophan, tyrosine and phenylalanine by second derivative spectrophotometry. Arch. Biochem. Biophys. 277:324‐333.
   Savitzky, A. and Golay, M.J.E. 1964. Smoothing and differentiation of data by simplified least squares procedures. Anal. Chem. 36:1627‐1639.
   Scopes, R.K. 1974. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59:277‐282.
   Servillo, L., Colonna, G., Balestrieri, C., Ragone, R., and Irace, G. 1982. Simultaneous determination of tyrosine and tryptophan residues in proteins by second‐derivative spectroscopy. Anal. Biochem. 126:251‐257.
   Steiner, J., Termonia, Y., and Deltour, J. 1972. omments on smoothing and differentiation of data by simplified least squares procedure. Anal. Chem. 44:1906‐1909.
   Timasheff, S.N. 1966. Turbidity as a criterion of coagulation. J. Colloid Interface Sci. 21:489‐497.
   Volkin, D.B. and Middaugh, C.R. 1992. The effect of temperature on protein structure. In Stability of Protein Pharmaceuticals, Part A: Chemical and Physical Pathways of Protein Degradation (T.J. Ahern and M.C. Manning, eds.) pp. 215‐247. Plenum Press, New York.
   Weidner, V.R., Mavrodineanu, R., Mielenz, K.D., Velapoldi, R.A., Eckerle, K.L., and Adams, B. 1985. Spectral transmittance characteristics of oxide in perchloric acid solution. J. Res. Natl. Bur.Stand. 90:115‐125.
   Wetlaufer, D.B. 1962. Ultraviolet spectra of proteins and amino acids. Adv. Protein Chem. 17:303‐390.
   Zavitsanos, P. and Goetz, H. 1991. The practical application of diode array UV‐visible detectors to high‐performance liquid chromatography analysis of peptides and proteins. In High‐Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis, and Conformation (C.T. Mant and R.S. Hodges, eds.) pp. 553‐562. CRC Press, Boca Raton, Fla.
Key References
   Cantor, C.R. and Schimmel, P.R. 1980. Absorption spectroscopy. In Biophysical Chemistry, Part II: Techniques for the Study of Biological Structure and Function, pp. 349‐408. W.H. Freeman, New York.
  Provides theoretical background and practical considerations in absorption spectroscopy.
   Wetlaufer, D.B. 1962. See above.
  Provides comprehensive discussion of factors involved in analysis of proteins using UV spectroscopy.
   Savitzky, A. and Golay, M.J.E. 1964. See above.
  Provides formulas for smoothing and differentiation of data.
   Steiner, J. et al. 1974. See above.
  Provides formulas for smoothing and differentiation of data.
   Levine, R.L. and Federici, M.M. 1982. See above.
  Describes an application of second‐derivative spectroscopy to analyze aromatic amino acid content employing a diode‐array spectrophotometer.
   Mach, H., Middaugh, C.R., and Lewis, R.V. 1992b. See above.
  Describes calculation of protein extinction coefficients based on sequence and provides a formula for light‐scattering correction at 280 nm.
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