Synthesis and Identification of Biologically Active Mono-Labelled FITC-Insulin Conjugate.
Blotting, Western
Cells, Cultured
Chromatography, High Pressure Liquid
/ methods
Chromatography, Reverse-Phase
/ methods
Fluorescein-5-isothiocyanate
/ analogs & derivatives
Fluorescence
Glucose Transporter Type 4
/ metabolism
Humans
Hydrogen-Ion Concentration
Insulin
/ analogs & derivatives
Mass Spectrometry
Muscle Cells
/ drug effects
Muscle, Skeletal
/ cytology
Phosphates
Phosphorylation
Proto-Oncogene Proteins c-akt
/ metabolism
Signal Transduction
Biologically active FITC-insulin
FITC
FITC-labelled insulin
Fluorescent labelling
Insulin
Journal
Journal of fluorescence
ISSN: 1573-4994
Titre abrégé: J Fluoresc
Pays: Netherlands
ID NLM: 9201341
Informations de publication
Date de publication:
Mar 2022
Mar 2022
Historique:
received:
24
09
2021
accepted:
03
12
2021
pubmed:
11
1
2022
medline:
24
3
2022
entrez:
10
1
2022
Statut:
ppublish
Résumé
Fluorescently labelling proteins such as insulin have wide ranging applications in a pharmaceutical research and drug delivery. Human insulin (Actrapid®) was labelled with fluorescein isothiocyanate (FITC) and the synthesised conjugate identified using reverse phase high performance liquid chromatography (RP-HPLC) on a C18 column and a gradient method with mobile phase A containing 0.1% trifluoroacetic acid (TFA) in Millipore water and mobile phase B containing 90% Acetonitrile, 10% Millipore water and 0.1% TFA. Syntheses were carried out at varying reaction times between 4 and 20 h. Mono-labelled FITC-insulin conjugate was successfully synthesised with labelling at the B1 position on the insulin chain using a molar ratio of 2:1 (FITC:insulin) at a reaction time of 18 h and confirmed by electrospray mass spectroscopy. Reactions were studied across a pH range of 7-9.8 and the quantities switch from mono-labelled to di-labelled FITC-insulin conjugates at a reaction time of 2 h (2:1 molar ratio) at pH > 8. The conjugates isolated from the studies had biological activities in comparison to native insulin of 99.5% monoB1, 78% monoA1, 51% diA1B1 and 0.06% triA1B1B29 in HUVEC cells by examining AKT phosphorylation levels. MonoB1 FITC-insulin conjugate was also compared to native insulin by examining cell surface GLUT4 in C2C12 skeletal muscle cells. No significant difference in the cellular response was observed for monoB1 produced in-house compared to native insulin. Therefore mono-labelled FITC-insulin at the B1 position showed similar biological activity as native insulin and can potentially be used for future biomedical applications.
Identifiants
pubmed: 35006485
doi: 10.1007/s10895-021-02867-1
pii: 10.1007/s10895-021-02867-1
doi:
Substances chimiques
Glucose Transporter Type 4
0
Insulin
0
Phosphates
0
SLC2A4 protein, human
0
insulin, fluorescein-isothiocyanated-
0
Proto-Oncogene Proteins c-akt
EC 2.7.11.1
Fluorescein-5-isothiocyanate
I223NX31W9
sodium phosphate
SE337SVY37
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
569-582Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.
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