scholarly journals N-terminal sequence analysis of polypeptide at the picomole level

1981 ◽  
Vol 199 (3) ◽  
pp. 557-564 ◽  
Author(s):  
J Y Chang
Keyword(s):  
High Pressure ◽  
Sequence Analysis ◽  
Visible Region ◽  
Reversed Phase ◽  
Complete Sequence ◽  
Complete Analysis ◽  
Phenyl Isothiocyanate ◽  
Terminal Sequence ◽  
Baseline Noise ◽  
Liquid Chromatographic

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.

scholarly journals The catalytic chain of human complement subcomponent C1. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments

1982 ◽  
Vol 201 (1) ◽  
pp. 49-59 ◽  
Author(s):  
G J Arlaud ◽  
J Gagnon ◽  
R R Porter
Keyword(s):  
High Pressure ◽  
Amino Acid ◽  
Gel Filtration ◽  
Gel Permeation Chromatography ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Serine Proteinases ◽  
Human Complement ◽  
Terminal Sequence ◽  
Cnbr Cleavage

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the ‘charge-relay’ system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the ‘histidine loop’, a disulphide bond that is present in all other known serine proteinases.

scholarly journals A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore

1983 ◽  
Vol 211 (1) ◽  
pp. 163-171 ◽  
Author(s):  
J Y Chang ◽  
R Knecht ◽  
D G Braun
Keyword(s):  
High Pressure ◽  
Light Chain ◽  
Visible Region ◽  
High Sensitivity ◽  
Thiol Group ◽  
Reversed Phase ◽  
New Method ◽  
Kappa Light Chain ◽  
Selective Isolation ◽  
Specific Labelling

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4′-iodoacetamide and 4-dimethylaminoazobenzene-4′-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.

Measurement of Nucleotide Pools in Platelets Using High Pressure Liquid Chromatography

1977 ◽  
Vol 38 (04) ◽  
pp. 0990-1001 ◽  
Author(s):  
Leo D’Souza ◽  
Helen I. Glueck
Keyword(s):  
High Pressure ◽  
Cyclic Nucleotides ◽  
Platelet Rich Plasma ◽  
Ammonium Phosphate ◽  
Single Step ◽  
Complete Analysis ◽  
Chromatographic Technique ◽  
Nucleotide Pools ◽  
Liquid Chromatographic ◽  
Camp And Cgmp

SummaryWe have devised an improved high pressure liquid chromatographic technique whereby serotonin, nucleosides, cyclic nucleotides, namely cAMP and cGMP, and 5’ mono-, 5’ di-, and 5’ tri-nucleotides can be analyzed. The cyclic nucleotides have been measured in pico molar quantities. All nucleotides can be quantitated in a single step separation in 75 min using a 0.0015 M phosphoric acids vs. 1 M pH 4.8 ammonium phosphate gradient. 5/10 ml of platelet-rich plasma furnishes an adequate sample for complete analysis. Nucleotide levels in platelets from 16 normal donors expressed in 1011 platelets are as follows: cAMP, 6.32 (4.15) nanomoles and AMP, 0.32(0.14); ADP, 2.48 (0.67); ATP 3.78 (0.68); GDP 0.38 (0.07) and GTP, 0.45 (0.07) micromoles. ADP and ATP values are lower than those previously published. However, the total nucleotide level approaches published values.Upon aggregation with thrombin, approximately 50% of ADP and 40 % ATP is released. Release is complete by 2 min. Thrombin is the most potent releasing agent with collagen and ADP occupying an intermediate role and epinephrine being the least effective. Upon aggregation cyclic AMP levels diminish along with other nucleotides. Patients with asthma showed depression of ADP, ATP, GDP and GTP levels.

High Pressure Liquid Chromatographic Determination of Chloramine-T in Food

1979 ◽  
Vol 62 (5) ◽  
pp. 1087-1091
Author(s):  
Paul R Beljaars ◽  
Theo M M Rondags
Keyword(s):  
High Pressure ◽  
Ice Cream ◽  
Hplc Method ◽  
Complete Analysis ◽  
High Pressure Liquid Chromatographic ◽  
Digital Integrator ◽  
Relative Standard ◽  
Chloramine T ◽  
Minced Meat ◽  
Liquid Chromatographic

Abstract A high pressure liquid chromatographic (HPLC) method for determining chloramine-T (N-chloro-N-sodium-p-toluenesulfonamide) in foods such as ice cream, minced meat, and shrimp is described. Deproteinized samples are treated with sulfite to convert chloramine-T to p-toluenesulfonamide (p-TSA) and extracted, and HPLC analysis is performed on concentrated extracts. Sample extracts are chromatographed on a reverse phase 10 μm Lichrosorb RP-18 column with acetonitrile-water (10+90), and quantitated by an ultraviolet detector (220 nm) and digital integrator. The relationship between recorded peak area and concentration was linear to 0.600 μg p-TSA/μL. The detection limit was 2.5 ng p-TSA/μL, corresponding to a chloramine-T concentration of 0.8 mg/kg in samples. Recoveries of added chloramine-T were 88% for ice cream, 73% for minced meat, and 51% for shrimp. Precision data indicate a relative standard deviation of 1.54 and 2.14% for the complete analysis of ice cream with levels of 15 and 63 mg chloramine-T/ kg (n = 5 determinations), respectively. The HPLC method was applied to chloramine-T determinations in 62 ice cream, 25 minced meat, and 25 shrimp samples.

Liquid-chromatographic quantification of plasma phenylalanine, tyrosine, and tryptophan.

1981 ◽  
Vol 27 (1) ◽  
pp. 146-148 ◽  
Author(s):  
L M Neckers ◽  
L E Delisi ◽  
R J Wyatt
Keyword(s):  
Amino Acids ◽  
High Pressure ◽  
Liquid Chromatography ◽  
Fluorescence Detection ◽  
Mobile Phase ◽  
Reversed Phase ◽  
Plasma Phenylalanine ◽  
Liquid Chromatographic ◽  
Phase Column ◽  
Reversed Phase Column

Abstract Phenylalanine, tyrosine, and tryptophan are isolated and quantified by "high-pressure" liquid chromatography, with fluorescence detection. An isocratic mobile phase and reversed-phase column are used to provide rapid and reproducible measurement of these amino acids in as little as 1 to 2 microL of human plasma.

A simple liquid-chromatographic method applied to determine caffeine in plasma and tissues.

1988 ◽  
Vol 34 (11) ◽  
pp. 2345-2348 ◽  
Author(s):  
I Biaggioni ◽  
S Paul ◽  
D Robertson
Keyword(s):  
High Pressure ◽  
Extraction Method ◽  
Mobile Phase ◽  
Chromatographic Method ◽  
Solid Phase ◽  
Reversed Phase ◽  
Simple Liquid ◽  
Liquid Chromatographic Method ◽  
The Mean ◽  
Liquid Chromatographic

Abstract In this simple, reliable assay for quantifying caffeine in plasma and tissues, methylxanthines are first partly purified from plasma and acid extracts of tissue by passage through solid-phase columns. The ease of this extraction method permits a relatively large number of samples to be processed daily. Quantification is by reversed-phase high-pressure liquid chromatography (mobile phase: acetic acid/acetonitrile/water, 2/6/92 by vol). Caffeine is eluted in 20 min. The reliability of this method allows its automation. This method has been adapted to measure caffeine in brain and kidney extracts and in as little as 10 microL of plasma. After 10 days of oral administration of caffeine (1 g/L, in drinking water) to six rats, the mean (+/- SEM) concentrations of caffeine in plasma, brain, and kidney were 18.6 +/- 6.0 micrograms/mL, 16.2 +/- 1.5 micrograms/g, and 18.9 +/- 2.0 micrograms/g, respectively. Correlations were linear between concentrations of caffeine in plasma and brain (r = 0.86) and between concentrations in plasma and kidney (r = 0.91). This method should be useful in studying the effects and mechanisms of actions of methylxanthines.

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