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Assay development for lysyl hydroxylase
Title
Assay development for lysyl hydroxylase.
Creator
Patel, Deepak A., Florida Atlantic University, Fields, Gregg B.
Abstract/Description
Hydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the...
Show moreHydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the corresponding hydroxylated (hydroxylysine-containing) peptide] using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. A new method for efficient separation of lysine- from hydroxylysine-containing peptides by HPLC has been developed in both organic phase (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). These reagents have been used to derivatize peptides prior to HPLC analysis. The products (di- and tetra-substituted lysine- and hydroxylysine-containing peptides) have been fully separated by HPLC and their structure confirmed by MALDI-TOF MS analysis. Efficient separation of derivatized peptides will allow for the convenient and rapid measurement of LH activity by HPLC methods.
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Date Issued
2006
PURL
http://purl.flvc.org/fcla/dt/13384
Subject Headings
Biological transport, Proteins--Metabolism, Peptides--Analysis, Coenzymes, Bioorganic chemistry
Format
Document (PDF)
Study of Cell Penetrating Peptide Uptake and Cancer Cell Discrimination with Raman Spectroscopy and Microscopy
Title
Study of Cell Penetrating Peptide Uptake and Cancer Cell Discrimination with Raman Spectroscopy and Microscopy.
Creator
Cosme, Patrick Jason, Terentis, Andrew C., Florida Atlantic University, Charles E Schmidt College of Science, Department of Chemistry and Biochemistry
Abstract/Description
Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs,...
Show moreCell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells CRM’s capability to discriminate control and treated cell groups was verified by principal component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100% accurate We’ve determined that at a concentration of 20 μM TP enters cells through a non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and results in a significant increase in cellular stiffness Our work provides the first direct evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was examined by CRM to determine the potential role of cyclic structure on cellular uptake The peptide, along with monocyclic and linear analogs was heavy isotope labeled and incubated with mammalian cells at numerous concentrations and timespans Our work is the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have been linked to artificial cellular uptake We demonstrate herein the absence of any CRM detectable uptake, providing the first evidence that SFTI-1 may not be a CPP Finally, CRM was applied to the discrimination of normal and basal cell carcinoma cells obtained from the same donor The use of patient matched cells avoids the normal biochemical variations that exist among individuals, ensuring that discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by PCA and LDA, were capable of spectral discrimination with 100% accuracy Major differences in the cancerous cells were an increase in lipids and nucleic acids, and an overall decrease in protein We also demonstrate an enhancement in Raman signal through the use of an aluminum foil substrate, providing a practical approach for measuring cells with thin morphologies
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Date Issued
2016
PURL
http://purl.flvc.org/fau/fd/FA00004756
Subject Headings
Peptides--Analysis, Peptides--Therapeutic use, Peptides--Physiological transport, Cellular signal transduction, Raman spectroscopy, Infrared spectroscopy
Format
Document (PDF)