An improved fluorogenic substrate for the alternative complement pathway C3/5 converting enzyme CVFBb.

J C Gutierrez; O Götze; L H Caporale

doi:10.1016/0167-4838(83)90200-5

Home/Archive/PubMed

PubMed · 6552199

An improved fluorogenic substrate for the alternative complement pathway C3/5 converting enzyme CVFBb.

J C Gutierrez·O Götze·L H Caporale

View original source ↗Export metadata ↓

Abstract

By lengthening the sequence of a previously available tripeptide to a pentapeptide, we were able to increase the specificity of the substrate for the complement enzyme CVFBb (EC 3.4.21.47) over Factor Xa (EC 3.4.21.6). This increase in specificity was achieved by both an increase in the kcat/Km for CVFBb for the longer substrate compared to the original substrate, and a decrease in the kcat/Km for Factor Xa. The new substrate, Boc-Nle-Gln-Leu-Gly-Arg-amino methyl coumarin (AMC) was synthesized by coupling Boc-Nle-Gln-Leu-Gly to Arg-AMC in solution. p-Toluenesulfonic acid was added to the coupling mixture to improve the solubility of the arginine derivative and avoid the need for covalent protection.

Explore related subjects

Chromatography, High Pressure Liquid Complement Activation Complement Factor B Complement Pathway, Alternative Coumarins Enzyme Precursors Factor X Factor Xa Fluorescent Dyes Kinetics Oligopeptides Substrate Specificity Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

J C Gutierrez, O Götze, L H Caporale. 1983-05-18. An improved fluorogenic substrate for the alternative complement pathway C3/5 converting enzyme CVFBb.. https://doi.org/10.1016/0167-4838(83)90200-5

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed1997

Redox potentials of glutaredoxins and other thiol-disulfide oxidoreductases of the thioredoxin superfamily determined by direct protein-protein redox equilibria.

Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX1X2C). Standard state redox potential (E degrees ') values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E degrees ' resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E degrees ' values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E degrees ' = -233 and -198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E degrees ' values from a number of proteins ranging from -270 mV for E. coli Trx to -124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as -240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.

Chromatography, High Pressure Liquid↗

PubMed1997

Information transfer from DNA to peptide nucleic acids by template-directed syntheses.

Peptide nucleic acids (PNAs) are analogs of nucleic acids in which the ribose-phosphate backbone is replaced by a backbone held together by amide bonds. PNAs are interesting as models of alternative genetic systems because they form potentially informational base paired helical structures. Oligocytidylates have been shown to act as templates for formation of longer oligomers of G from PNA G2 dimers. In this paper we show that information can be transferred from DNA to PNA. DNA C4T2C4 is an efficient template for synthesis of PNA G4A2G4 using G2 and A2 units as substrates. The corresponding synthesis of PNA G4C2G4 on DNA C4G2C4 is less efficient. Incorporation of PNA T2 into PNA products on DNA C4A2C4 is the least efficient of the three reactions. These results, obtained using PNA dimers as substrates, parallel those obtained using monomeric activated nucleotides.

Chromatography, High Pressure Liquid↗

PubMed1997

Development of an assay for the simultaneous determination of sildenafil (Viagra) and its metabolite (UK-103,320) using automated sequential trace enrichment of dialysates and high-performance liquid chromatography.

The development of the ASTED (automated sequential trace enrichment of dialysates) system to prepare plasma samples prior to high-performance liquid chromatography (HPLC) of sildenafil and its demethylated metabolite (UK-103,320) is described. Investigations to elucidate potential pitfalls of the ASTED on-line sample preparation system prior to separation of the analytes by HPLC are presented. The procedure is shown to be selective for sildenafil and UK-103,320, and linear over the range 1.00-250 ng/ml. The intra-batch imprecision (C.V.) of the method at plasma analyte concentrations of 1.00, 5.00, 50.0 and 200 ng/ml was 11.2, 3.10, 1.50, and 1.30%, respectively, and the corresponding inter-batch imprecision was estimated to be 13.5, 7.09, 3.69, and 5.43%. At these plasma analyte concentrations the overall inaccuracy (% bias) of the procedure ranged from 3.6 to 8.4%. The method showed similar assay performance for the estimation of the metabolite, UK-103,320. The application of the assay to a pharmacokinetic investigation during a clinical study is presented.

Chromatography, High Pressure Liquid↗