Optimization of the conditions for biuret complex formation for the determination of peptides by capillary electrophoresis with ultraviolet detection

Gawron, Andrew J.; Lunte, Susan M.

doi:10.1002/1522-2683(20000601)21:10<2067::aid-elps2067>3.0.co;2-5

Cited by 15 publications

(18 citation statements)

References 16 publications

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“…4 indeed gives a picture of b 2 -m complexed with Cu 11 at concentration values close to saturation: the experimentally measured effective mobility of I 2 peak at 15 mM Cu 11 is -5.24 6 0.0466 10 -5 cm 2 V -1 s -1 , very close to the complex mobility calculated by the y-reciprocal linearization plot (-5.191 6 0.017610 -5 cm 2 V -1 s -1 ). Furthermore, the increased peak height is possibly the result of a larger UV response upon Cu 11 complexation [44]. That the affinity constants of Cu 11 for I 2 calculated in this study, both with linear and non linear fitting, would result very close to the value recently found by fluorescence spectroscopy for the native b 2 -m [24] is certainly a tricky point that will deserve further consideration and investigation.…”

Section: Discussionmentioning

confidence: 85%

Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

et al. 2002

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Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.

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Section: Discussionmentioning

confidence: 85%

Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

et al. 2002

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“…Optimization of the complexation reaction was previously investigated by our group by comparing modified/oncapillary, precapillary, on-capillary, and pre/on-capillary complexation [28]. It was shown that on-capillary complexation had significant advantages over the other complexation methods.…”

Section: Optimization Of Biuret Conditionsmentioning

confidence: 99%

“…Initial reports for copper-peptide complexes with UV detection used a detection wavelength of 214 nm [28,34]. However, we found that a better signal-to-noise ratio was obtained at 200 nm for the detection of copperangiotensin peptide complexes, and this was employed for all further studies.…”

Section: Optimization Of Biuret Conditionsmentioning

confidence: 99%

“…However, Gawron et al [28] showed that the addition of copper to the run buffer actually improved the separation of Leu-enkephalin and its metabolites. This was also found to be true for angiotensin and its metabolites.…”

Section: Separation and Detection On Angiotensin And Metabolites By Cmentioning

confidence: 99%

“…Recently, our group has shown that complexes could be formed on-capillary [28][29][30]. On-capillary complexation is useful for the analysis of biological samples, because complexation does not result in sample dilution.…”

Section: Introductionmentioning

confidence: 99%

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Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection

2002

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The use of capillary electrophoresis (CE) with on-capillary Cu(II) complexation for the determination of angiotensin and its metabolites is described. The resulting copper-peptide complexes can be detected using either UV or electrochemical (EC) detection. Optimal reaction and separation conditions for the angiotensin peptides were first determined using CE with UV detection. With UV detection, the limit of detection (signal-to noise ratio S/N = 3) for native angiotensin II was 18 microM, while the limit of detection (LOD) obtained for the copper-angiotensin II complex is 2 microM. CE with EC detection was then evaluated, yielding significantly lower LODs--2 microM for native angiotensin II and 200 nM for the copper-angiotensin II complex. The addition of copper to the run buffer improved the separation and sensitivity for both CE-UV and CE-EC detection. The method was demonstrated by monitoring the conversion of angiotensin I to angiotensin II in plasma via angiotensin-converting enzyme (ACE) and subsequent inhibition of ACE by captopril.

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Detection of neuropeptides using on-capillary copper complexation and capillary electrophoresis with electrochemical detection

Gawron

Lunte

2000

Electrophoresis

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Capillary electrophoresis with electrochemical detection using a carbon fiber electrode in conjunction with on-capillary copper complexation was evaluated for the determination of peptides in standard and biological matrices. Peptides composed of 2-10 amino acids were investigated. A comparison was made between the responses obtained for peptides containing the oxidizable residue tyrosine and those obtained for their respective copper complexes. Electrochemical detection of non-tyrosine-containing peptides and a cyclic peptide was also demonstrated. A separation of leucine (Leu)-enkephalin and five metabolites was developed and then used for the investigation of Leu-enkephalin metabolism in plasma. The appearance of the des-tyrosine (des-Tyr) Leu-enkephalin, which cannot be detected directly at a carbon electrode, was monitored using the on-capillary complexation technique. Direct injection of the plasma sample was possible using this methodology.

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Optimization of the conditions for biuret complex formation for the determination of peptides by capillary electrophoresis with ultraviolet detection

Cited by 15 publications

References 16 publications

Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

Capillary electrophoresis investigation of a partially unfolded conformation of β2-microglobulin

Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection

Detection of neuropeptides using on-capillary copper complexation and capillary electrophoresis with electrochemical detection

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