scispace - formally typeset
Search or ask a question
Journal ArticleDOI

Calculation of protein extinction coefficients from amino acid sequence data

01 Nov 1989-Analytical Biochemistry (Anal Biochem)-Vol. 182, Iss: 2, pp 319-326
TL;DR: In this article, a method for calculating accurate molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition, was presented, and the method was calibrated against 18 "normal" globular proteins.
About: This article is published in Analytical Biochemistry.The article was published on 1989-11-01. It has received 5077 citations till now. The article focuses on the topics: Globular protein & Molar absorptivity.
Citations
More filters
Book ChapterDOI
TL;DR: Details are given about protein identification and analysis software that is available through the ExPASy World Wide Web server and the extensive annotation available in the Swiss-Prot database is used.
Abstract: Protein identification and analysis software performs a central role in the investigation of proteins from two-dimensional (2-D) gels and mass spectrometry. For protein identification, the user matches certain empirically acquired information against a protein database to define a protein as already known or as novel. For protein analysis, information in protein databases can be used to predict certain properties about a protein, which can be useful for its empirical investigation. The two processes are thus complementary. Although there are numerous programs available for those applications, we have developed a set of original tools with a few main goals in mind. Specifically, these are: 1. To utilize the extensive annotation available in the Swiss-Prot database wherever possible, in particular the position-specific annotation in the Swiss-Prot feature tables to take into account posttranslational modifications and protein processing. 2. To develop tools specifically, but not exclusively, applicable to proteins prepared by two dimensional gel electrophoresis and peptide mass fingerprinting experiments. 3. To make all tools available on the World-Wide Web (WWW), and freely usable by the scientific community. In this chapter we give details about protein identification and analysis software that is available through the ExPASy World Wide Web server.

8,007 citations

Journal ArticleDOI
C. N. Pace1, Felix Vajdos1, L. R. Fee1, Gerald R. Grimsley1, T Gray1 
TL;DR: The studies reported here suggest that the Edelhoch method is the best method for measuring ε for a protein, which can best be predicted with this equation.
Abstract: The molar absorption coefficient, E, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring E for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:3193261 and is based on data from Edelhoch [1967, Biochemistry 6:1948-19541.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average E values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the E values in water, propanol, 6 M guanidine hydrochloride (GdnHCI), and 8 M urea have been measured. For Trp, the average E values for the proteins are less than the E values measured in any of the solvents. For Tyr, the average E values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured t values for 80 proteins, the t at 280 nm of a folded protein in water, t(280), can best be predicted with this equation:

3,718 citations

Journal ArticleDOI
TL;DR: The basis of the CD approach and its application to the study of proteins, and clear guidelines on how reliable data can be obtained and analysed are presented.

3,023 citations

Journal ArticleDOI
TL;DR: Six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria are cloned, two of which have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths.
Abstract: We have cloned six fluorescent proteins homologous to the green fluorescent protein (GFP) from Aequorea victoria. Two of these have spectral characteristics dramatically different from GFP, emitting at yellow and red wavelengths. All the proteins were isolated from nonbioluminescent reef corals, demonstrating that GFP-like proteins are not always functionally linked to bioluminescence. The new proteins share the same beta-can fold first observed in GFP, and this provided a basis for the comparative analysis of structural features important for fluorescence. The usefulness of the new proteins for in vivo labeling was demonstrated by expressing them in mammalian cell culture and in mRNA microinjection assays in Xenopus embryos.

1,960 citations

Journal ArticleDOI
TL;DR: This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages, which are the direct sensors of inducers of the phase 2 system.
Abstract: Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [3H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C257, C273, C288, and C297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

1,812 citations

References
More filters
Journal Article
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.

289,852 citations

Journal ArticleDOI
TL;DR: This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr with little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose.

225,085 citations

Journal ArticleDOI
TL;DR: A procedure is presented which strongly reduces or elimi- nates these interactions, normalizes their absorption, and consequently permits a more precise analysis of tryptophan and tyrosine in proteins.
Abstract: A rapid method for the determination of tryptophan in proteins is presented. It is based on ab- sorbance measurements at 288 and 280 mp of the protein dissolved in 6 M guanidine hydrochloride. Blocked tryptophanyl (N-acetyl-L-tryptophanamide) and tyrosyl (glycyl-L-tyrosylglycine) compounds were selected as C urrent methods of protein amino acid analysis do not give quantitative values for tryptophan and conse- quently the amino acid compositions, which are other- wise complete, fail to report tryptophan values. The principal reason for this situation is that the standard procedure of protein hydrolysis in strong acid results in the destruction of tryptophan (Hill, 1965). Therefore a second procedure is required to measure tryptophan. Alkaline hydrolysis is less destructive but does not give quantitative recoveries generally (Spies and Chambers, 1949). Enzymatic hydrolysis of proteins can give quanti- tative yields of tryptophan but this method may not be generally valid (Hill and Schmidt, 1962). The hydrolytic problem can be circumvented by meas- uring tryptophan in the intact protein. A chemical method has been developed which has not been exploited adequately (Spies and Chambers, 1948, 1949). On the other hand, considerable effort has been expended in developing absorption spectroscopic procedures to measure tryptophan and tyrosine in unhydrolyzed pro- teins. Holiday (1936) and Goodwin and Morton (1946) have measured the absorption of proteins in 0.1 M NaOH and computed their tryptophan and tyrosine contents based on comparison with the absorption of the two amino acids. A modification of these techniques has been presented by Bencze and Schmid (1957). The pre- ceding three methods do not give quantitative results. The behavior of the chromophores has not been nor- malized and the two models, i.e., tryptophan and tyro- sine, are not completely adequate. A procedure is sug- gested in this report which strongly reduces or elimi- nates these interactions, normalizes their absorption, and consequently permits a more precise analysis of tryptophan and tyrosine in proteins.

3,323 citations

Book ChapterDOI
TL;DR: This chapter deals with the absorption spectra of proteins and amino acids, principally derived from the study of homogeneous absorbing systems, in which the inhomogeneity is finer in grade by several orders than the dimensions of the exploring light beam.
Abstract: Publisher Summary This chapter deals with the absorption spectra of proteins and amino acids The colored proteins are conjugated proteins in which the protein carrier is colorless This transparency of protein solutions extends into the ultraviolet region of the spectrum and many proteins do not absorb radiation of longer wavelength than 2500 Ǻ The essential protein fabric, consisting of a peptide chain in various forms, is not responsible for absorption at longer wavelengths In case of fibrous proteins, there is some evidence that the peptide fabric is responsible for absorption in this region Many proteins absorb in this region This absorption is due to the aromatic amino-acids present in the protein The advent of quantitative methods of spectrophotometry is the basis of a method of determining tyrosine and tryptophan in proteins The striking property of proteins is their transparency, indicating a high degree of electronic saturation The configurational stability of the protein molecule depends entirely on extra-valence forces and not on unsaturation, which would result in high absorption in the ultraviolet The absence of such rigidifying bonds endows the protein with its unique characters of plasticity, while the number-sequence of side chains gives its chemical constancy These two properties allow these molecules to be arranged in large polymorphic masses to form a matrix fabric of recurrent pattern in media, which are essentially aqueous The material reviewed is principally derived from the study of homogeneous absorbing systems, in which the inhomogeneity is finer in grade by several orders than the dimensions of the exploring light beam

944 citations

Journal ArticleDOI
TL;DR: The estimated ϵ205 is about three times better than using an average ϵ2051 mg/ml of 31 and approaches the range of experimental error inherent in any method of protein estimation.

688 citations