Identification of enzymatically produced peptide fragments by liquid chromatography and mass spectrometry

The qualitative and quantitative determination of peptide fragments of angiotensin I generated by rat lung dipeptidyl carboxypeptidase (angiotensin converting enzyme, EC 3.4.15.1) is described. Enzymatically formed peptide fragments, after derivatization with fluorescamine, were separated and isolated by reverse-phase high-performance liquid chromatography. The recovered fluorescamine derivative of histidyl-leucine was then further identified by mass spectrometry. It is anticipated that this approach would be widely applicable to other enzyme systems.     

Simultaneous analyses of phenethylamine, phenylethanolamine, tyramine and octopamine in rat brain using fluorescamine

A fluorometric method for the simultaneous analyses of phenethylamine, phenylethanolamine, tyramine and octopamine has been developed. The method involves ion-exchange chromatography, derivatization with fluorescamine, solvent extraction and then separation by thin-layer chromatography. The fluorescent spots are then quantitated by scanning. The detection limits of this method are about 10 pmoles for phenethylamine, phenylethanolamine and tyramine, and 20 pmoles for octopamine. The method was used for simultaneous analyses of putative neurotransmitter amines in whole rat brain.     

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 5 pmole for spermine and the others in 0.5 ml of serum, respectively.     

The use of fluorescamine as a detection reagent in protein microcharacterization

With recent advances in protein microchemistry, compatible methods for the preparation and quantitation of proteins and peptides are required. Fluorescamine, a reagent which reacts with primary amino groups has been used successfully to detect amino acids, peptides, and proteins in various micromethods. This article discusses these methods which include (1) amino acid analysis of protein and peptide hydrolysates with postcolumn fluorescamine derivatization; (2) purification and characterization of proteins and peptides by reversed-phase HPLC with postcolumn fluorescamine derivatization; (3) purification of peptides by two-dimensional chromatography and electrophoresis on thin-layer cellulose with fluorescamine staining; and (4) electroblotting of protein bands from SDS-PAGE to glass fiber filters and polyvinylidene difluoride (PVDF) membranes with fluorescamine staining. In addition, this article also compares a postcolumn fluorescamine detection system with a UV detection system in the applications of amino acid analysis and reversed-phase HPLC protein/peptide analysis.     

Determination of long-chain base in glycosphingolipids with fluorescamine

A method is described for determining the long-chain base content of glycosphingolipids after acid hydrolysis, using the new reagent fluorescamine. The reaction is sensitive and can be used to characterize or measure glycosphingolipids in quantities routinely separated by thin-layer chromatography.     

Pre-column derivatization with fluorescamine and high-performance liquid chromatographic analysis of drugs : Application to tocainide

The quantitative determination of tocainide, a new antiarrhythmic agent, by high-performance liquid chromatography (HPLC) is reported. The drug and a chemically similar internal standard were extracted from blood plasma with acetonitrile under salting-out conditions obtained by saturation of the aqueous medium with sodium chloride—sodium carbonate. The organic extract, without evaporation, was treated with borate buffer (pH 8.2) and fluorescamine. The resulting derivatives were chromatographed on an ODS reversed-phase column using a methanol—phosphate buffer (pH 7.0) mixture as mobile phase and were detected fluorometrically by monitoring the emission at 485 nm, with excitation at 395 nm. The intra-assay coefficients of variation were 3.0 and 4.3% for ten replicate 0.25 and 1.00 μg/ml samples, respectively, and the inter-assay coefficient of variation was 3.6% for ten replicate 1.00 μg/ml samples. The procedure is simple, rapid, sensitive, and specific. Several other drugs and drug metabolites also were derivatized with fluorescamine and chromatographed successfully. Pre-column derivatization with fluorescamine followed by HPLC with fluorometric detection may have significant advantages in drug analysis.     

A High Performance Liquid Chromatography Method for Measuring Brain Dihydroxyphenylalanine Levels and Dopamine Synthesis Rates

Abstract: A high performance liquid chromatography method by which one can detect as little as 45 pmol of dihydroxyphenylalanine in brain tissue is described. Dihydroxyphenylalanine was reacted with fluorescamine and was quantitated, after chromatography, by fluorescence spectroscopy. The steady state levels of dihydroxyphenylalanine in the mouse striatum were found to be below 90 pmol/g, but the dihydroxyphenylalanine levels were rapidly increased by inhibition of aromatic amino acid decarboxylase. The method described was utilized to demonstrate the effects of pharmacologic agents on dihydroxyphenylalanine synthesis in mouse striatum.     

Determination of milnacipran, a serotonin and noradrenaline reuptake inhibitor, in human plasma using liquid chromatography with spectrofluorimetric detection

Milnacipran is an antidepressant drug belonging to the class of serotonin and noradrenaline reuptake inhibitors. A sensitive high performance liquid chromatographic during the development method coupled with a fluorimetric detection was set up, validated and then used routinely of the drug. After liquid-liquid extraction, milnacipran and its internal standard were analyzed by reversed-phase liquid chromatography (LC). The drug was derivatized with fluorescamine for fluorescence detection. The identity of the liquid chromatography peaks was controlled using mass spectrometry. The assay linearity was validated up to 1000 ng/ml. The limit of quantification was set at 5 ng/ml. Precision values (relative standard deviations) were lower than 5.4%, whereas the mean accuracy was higher than 95%. The extraction recoveries were higher than 70% for both milnacipran and the internal standard. In clinics, the LC-fluorescence method was routinely used to investigate the pharmacokinetics of milnacipran in patients and proved to be robust and capable of quantifying milnacipran in plasma for at least 36 h (four- to five-fold the elimination half-life).     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

Profiling of urinary medium-sized peptides in normal and uremic urine by high-performance liquid chromatography

This report describes the profiling of medium-sized peptides in both normal and uremic urine by ion-pair reversed-phase high-performance liquid chromatography using an acetonitrile—heptafluorobutyric acid solvent system as eluent. Several medium-sized peptide peaks could be detected in both normal and uremic urine at low picomole level by using post-column fluorescence derivatization with fluorescamine. Contrary to expectation, uremic urine contained slightly larger amounts of medium-sized peptides compared with normal urine.     

Characterization of proteins and peptides by high-performance liquid chromatography and fluorescence monitoring of their tryptic digests.

Proteins and peptides can be characterized and compared at the subnanomole level by treatment with trypsin followed by high-performance liquid chromatography on reverse-phase partition columns. A fluorescamine monitoring system automatically analyzes a portion of the column effluent while the remainder can be collected for further studies. The method has already been used for characterization of rat β-endorphin and a protein which cross-reacts with antiserum prepared against prolyl hydroxylase.     

Bioorganic chemistry of the purple membrane ofHalobacterium halobium — Chromophore and apoprotein modified bacteriorhodopsins

Iodophenyl and anthryl retinal analogues have been synthesized. Thetrans-isomers have been isolated and purified by high pressure liquid chromatography. The purified isomers have been further characterized by nuclear magnetic resonance and ultraviolet-visible spectroscopy. Incubation of these retinal analogues with apoprotein (bacterioopsin), isolated from the purple membrane ofHalobacterium halobium gave new bacteriorhodopsin analogues. These analogues have been investigated for their absorption properties and stability. The iodophenyl analogue has been found to bind to bacterioopsin rapidly. The pigment obtained from this analogue showed a dramatically altered opsin shift of 1343 cm^-1. The anthryl analogue based bacteriorhodopsin, however, showed an opsin shift of 3849 cm^-1. It has been found that bacteriorhodopsin is quite unrestrictive in the ionone ring site. The apoprotein seems to prefer chromophores that have the ring portion co-planar with the polyene side chain. The purple membrane has also been modified by treatment with fluorescamine, a surface active reagent specific for amino groups. Reaction under controlled stoichiometric conditions resulted in the formation of a modified pigment. The new pigment showed a band at 390 nm—indicative of fluorescamine reaction with amino group (s) of apoprotein-besides retaining its original absorption band at 560 nm. Analysis of the fluorescamine modified bacteriorhodopsin resulted in the identification of lysine 129 as the modified amino acid residue. Fluorescamine-modified-bacteriorhodopsin suspension did not release protons under photolytic conditions. However, proteoliposomes of fluorescamine-modified-bacteriorhodopsin were found to show proton uptake, though at a reduced rate. Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.     

Fluorescent thin-layer peptide mapping for protein identification and comparison in the subnanomole range

Conditions and simple precautions are presented for carrying out highly reproducible and sensitive peptide mapping by thin-layer chromatography and subsequent electrophoresis of subnanomole amounts of tryptic digest on silica gel G or GHL plates. The fluorogenic reagent “fluorescamine” is employed for visualization under long-wavelength ultraviolet illumination. Permanent photorecording of high-contrast images, using readily available filters, is substituted for subjective hand scoring of plates. Contrast reversal is used to produce peptide maps suitable for half-tone reproduction.     

Single column analysis of amino acids in protein hydrolysates utilizing the fluorescamine reaction

A system is described for the separation of the amino acids commonly found in protein hydrolysates at the picomole level using a single ion exchange column and for their quantitation by the fluorescamine (4-phenylspiro[furan-2 (3H),1′-phthalan]-3,3′-dione) reaction. Three sodium citrate buffers were required for the separation of the amino acids with an analysis time of approximately 3 hr. The amino acids in 1 μg of hydrolyzed bovine serum albumin were separated using a single ion exchange column and were detected in the effluent from the column by the fluorescamine assay. The results were compared with those obtained using a commercial amino acid analyzer and 150 μg of hydrolyzed bovine serum albumin. The chromatogram produced by the more sensitive analyzer utilizing the fluorescamine reaction to detect the amino acids compared favorably with the chromatogram produced by the commercial analyzer utilizing the ninhydrin reaction with the exception that the proline peak was missing. Proline and hydroxyproline fail to yield fluorescence on reaction with fluorescamine unless converted from imines to primary amines.     

Membrane Studies in Huntington's Disease: Steady State and Time-Dependent Fluorescence Spectroscopy of Intact Lymphocytes

Abstract: Recent evidence suggests that a membrane abnormality, expressed in peripheral tissues such as the lymphocyte, may be present in Huntington's disease (HD). Both steady state and time-dependent fluorescence spectroscopic methods were performed on lymphocytes from patients with HD and from age- and sex-matched controls. Lymphocyte membrane dynamics were studied, using fluorescence probes with known specificity for certain membrane areas. These probes included 4-phenylspiro(furan-2(3H)-1'-phthalan)-3,3'-dione (fluorescamine), which binds to surface membrane primary amines, 1–8-anilinonaphthalene sulfonate (1,8-ANS), which inserts at the aqueous-hydrocarbon interface, and 12(9) anthroyl stearate (12(9)AS), which inserts deep in the hydrocarbon core. Steady state fluorescence studies, using fluorescamine, 1–8 ANS, or 12(9)AS, revealed no significant difference between intact HD and control lymphocytes. Time-dependent energy-transfer polarization studies for fluorescamine (tryptophan → fluorescamine) did, however, reveal a slower time decay of I₁/I for intact HD lymphocytes as compared with controls. This time-dependent difference may relate to alterations in translational (lateral) and angular mobilities of membrane donors (tryptophan) relative to acceptors (fluorescamine) in intact HD lymphocytes. Such observations support the concept of a membrane abnormality in HD.     

Protamine, a substrate for thrombolytic agents and enzymes of similar specificity.

Protamine, a highly purified basic polypeptide of 4000 molecular weight containing 80–85% arginine, is a useful substrate for the assay of plasmin, activated plasminogen, and enzymes of similar specificity, e.g., urokinase, coagulation factor Xa, trypsin, and thrombin, and is also an excellent secondary substrate for activator assays of urokinase and streptokinase. The assays were performed manually, and also automated procedures for continuous multiple sample analyses were used. The relative sensitivities for various plasmin-like enzymes were: trypsin > plasmin > urokinase > factor Xa > thrombin. Using protamine with manual assay procedures, the amino-terminal groups of the enzyme-degraded protamine digestion products were detected and quantitated by the colorimetric ninhydrin or the fluorometric fluorescamine procedures, and using protamine with an automated system the ninhydrin method was used. Assigning the CTA casein assay for plasmin a nominal sensitivity of 0.1 (for 0.1 CTA unit of plasmin), the sensitivities of the various assay methods were casein, automated protamine, and manual protamine with ninhydrin, 0.1; manual protamine-trichloroacetic acid with fluorescamine, 0.005; and manual protamine direct fluorescamine, 0.0005. A unit of plasmin, based on the uptake of 1 μequiv base/min during hydrolysis of 0.4% protamine sulfate under standard conditions, is equal to approximately 1.7 × 10³ RFI units or 2.9 CTA units; or, 1 CTA unit of plasmin resulted in an average uptake of 0.346 μmol of base or equivalent bonds split per minute.     

A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol

An alternative primary amine assay utilizing 4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione (fluorescamine) is compared with the frequently applied trinitrobenzenesulfonic acid assay as a means of estimating protein primary amine modification with cyanuric chloride attachment of polyethylene glycol (PEG). The results of the two assays are compared for three different proteins and several advantages of the fluorescamine assay are emphasized. In particular, the fluorescamine assay was found to be unaffected by free PEG up to 0.08% in the assay mixture. The fluorescamine assay can be used to measure the partition coefficients of native or PEG-modified protein in two-phase aqueous polymer systems.     

High-performance liquid chromatographic method for determination of gentamicin in biological fluids

A selective and sensitive method for the determination of gentamicin in plasma and urine by high-performance liquid chromatography has been developed. Following deproteinization, the gentamicin is reacted with fluorescamine to produce a fluorescent derivative. This reaction mixture is directly chromatographed on a cation-exchange column using as mobile phase acetonitrile—phosphoric acid (7:3). The gentamicin components elute as a single peak. Using 0.1 ml of plasma, quantitation of gentamicin concentrations as low as 1 mg/l are possible. Possible interference from other aminoglycosides and antibiotics is discussed.     

Reaction of tryptophan,tryptamines, and some related indoles with fluorescamine: Unique fluorescence in strong acid

Tryptophan, peptides containing N-terminal tryptophan, tryptamine, and certain related compounds react with fluorescamine to form derivatives with uniquely high fluorescence in strong acid.     

Fluorometric assay of secondary amino acids

A method is described for the conversion of secondary amino acids to primary amines which can be assayed with fluorescamine (I). Secondary amino acids undergo oxidative decar?ylation when reacted with halogenating agents. The resulting imines are hydrolyzed to primary amines, which are subsequently allowed to react with fluorescamine (I) to yield fluorescent pyrrolinones (II). This reaction sequence provides an efficient fluorometric assay for secondary amino acids. Thus, the fluorescamine procedure is now applicable to the full array of natural amino acids.