Identification of Chemical Modification Sites on Metalloproteins by Capillary Electrophoresis

doi:10.1006/abio.1993.1285

Analytical Biochemistry

Volume 212, Issue 1, July 1993, Pages 24-27

https://doi.org/10.1006/abio.1993.1285 Get rights and content

Abstract

Capillary electrophoresis (CE) has been used to separate the peptides obtained from tryptic digestion of ruthenium-modified cytochrome c. The modified peptide was identified from a comparison of the elution profile and absorbance characteristics of the native and modified proteins. Automatic fraction collection on the CE instrument provides sufficient amounts of this modified peptide for amino acid sequencing. Capillary electrophoresis has also been used to monitor the modification reaction and to optimize the modification efficiency. The methodologies have been extended to myoglobin in order to monitor the modification of multiple surface sites.

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Cited by (7)

Ru(II)-diimine functionalized metalloproteins: From electron transfer studies to light-driven biocatalysis
2016, Biochimica et Biophysica Acta - Bioenergetics
Citation Excerpt :
However, despite recent progress, crystallization of macromolecules still remains a laborious process. Information on the selective covalent attachment can also be obtained from tryptic digest of the labeled metalloenzymes and analysis of the peptidic fragments [25,45]. This method was initially used to establish the labeling of ferricytochrome c with pentaamineruthenium(III) complexes.
The unique photochemical properties of Ru(II)-diimine complexes have helped initiate a series of seminal electron transfer studies in metalloenzymes. It has thus been possible to experimentally determine rate constants for long-range electron transfers. These studies have laid the foundation for the investigation of reactive intermediates in heme proteins and for the design of light-activated biocatalysts. Various metalloenzymes such as hydrogenase, carbon monoxide dehydrogenase, nitrogenase, laccase and cytochrome P450 BM3 have been functionalized with Ru(II)-diimine complexes. Upon visible light-excitation, these photosensitized metalloproteins are capable of sustaining photocatalytic activity to reduce small molecules such as protons, acetylene, hydrogen cyanide and carbon monoxide or activate molecular dioxygen to produce hydroxylated products. The Ru(II)-diimine photosensitizers are hence able to deliver multiple electrons to metalloenzymes buried active sites, circumventing the need for the natural redox partners. In this review, we will highlight the key achievements of the light-driven biocatalysts, which stem from the extensive electron transfer investigations. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.
Capillary electrophoresis of peptides in isoelectric buffers
1997, Journal of Chromatography A
Peptide maps in capillary zone electrophoresis are typically obtained in 80 mM phosphate buffer, pH 2.0, since at this pH value essentially all surface silanols of silica are protonated and thus one obviates to the need of chemically treating the inner capillary surface. When developing a peptide map of a tryptic digest of bovine β-casein under these conditions, good separations were obtained, but at the expense of a rather long running time (80 min) due to the low maximum voltages applicable (only 110 V/cm) under experimental conditions utilizing relatively large bore capillaries (100 μm I.D.) for increased sensitivity. When resorting to isoelectric aspartic acid as the sole buffering ion [pH=isoelectric point (pI)=2.77 at 25°C] much higher voltages could be applied (up to 800 V/cm) with a concomitant reduction of total running time (down to only 6–7 min). However, at this operative pH, strong adsorption of larger peptides to the capillary wall was noticed. This inconvenience was fully eliminated when finally adopting a buffer of the following composition: 50 mM Asp, pH 2.77, added with 0.5% hydroxyethylcellulose number-average molecular mass (M_n) of 27 000 and with 5% 2,2,2-trifluoroethanol. In this buffer, very high resolution could be achieved, due to the very low band spread at such greatly reduced running times and to the absence of wall adsorption. The most important parameter, when selecting amphoteric buffers, is their buffering power, which is a function of their pI−pK values. The buffering power, for isoelectric amino acids, is very strong for Lys and Asp (27 and 26 mequiv. l⁻¹ pH⁻¹, respectively, for 50 mM solutions), intermediate in the case of Glu (20), and rather minute for His and Arg (6 and 5.5, respectively). It is suggested that the minimum buffering power for a background electrolyte in CZE should be kept in the order of ≥10 mequiv. l⁻¹ pH⁻¹.
Isoelectric focusing as a tool for the investigation of post-translational processing and chemical modifications of proteins
1995, Journal of Chromatography A
It has been demonstrated that good agreement may be observed between computed and experimental isoelectric point (pI) values when proteins of known sequence are focused under denaturing conditions on immobilized pH gradient IPG slabs, at least in the pH range 4–7.5. Hence, discrepancies between expected and found in this experimental set-up may be reliably ascribed to some kind of post-transcriptional processing, or chemical modification, having taken place in the sample. This evaluation is made easier when the comparison is set between the pI of a parent molecule and that (or those) of one to several of its derivatives as resolved in a single experiment (for instance, as a spot row in two-dimensional maps); no previous knowledge is required in these cases about the amino acid composition of the primary structure. The effects on protein surface charge are discussed in this review mainly for two biologically relevant processes, glycosylation and phosphorylation. Then, the pI shifts are analysed for some protein modifications that may occur naturally but can also be artefactually elicited, such as NH₂ terminus blocking, deamidation and thiol redox reactions. Finally, carboxymethylation and carbamylation are used to exemplify chemical treatments often applied in connection with electrophoretic techniques and involving charged residues. Procedures to be applied in order to verify whether a given modification has occurred, and often relying on the focusing of a treated specimen, are detailed in each section. Numerical examples on model proteins are also discussed. As an important field of application of the above concepts may be genetic engineering, an exhaustive bibliographic list dealing with pI evaluation and structural assessment on recombinant proteins is included.
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Regular ArticleIdentification of Chemical Modification Sites on Metalloproteins by Capillary Electrophoresis

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Regular Article
Identification of Chemical Modification Sites on Metalloproteins by Capillary Electrophoresis