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Several excellent procedures for trapping tagged proteins have been devised, but many of these are expensive, cannot be used outside a limited pH range, fail to work in the presence of chaotropic agents, or are difficult to use. The chitin binding domain (CBD) of Bacillus circulans chitinase, which binds to chitin matrices prepared from inexpensive reagents isolated from crab shells, is an alternative tag that can be used under a variety of pH and denaturing conditions. Kits based on the interaction between the CBD and the chitin beads are available commercially. Here, we show that simultaneous treatment of microtiter plates with chitosan, a deacetylated form of chitin, and acetic anhydride produces a surface-bound film of chitin that also interacts tightly with the CBD. Chitin-coated microtiter well plates captured a CBD-tagged heterodimeric human glycoprotein hormone analog directly from mammalian cell culture media, even when present in trace amounts. Binding to the surface was stable in sodium dodecylsulfate and reversed only partially at low pH or in 8M urea at 37 degrees C. This technique appears well suited to surface attachment and permits biochemical or other analyses of molecules that can be tagged with a CBD.

Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10 11 molecules/mm 2 , which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.

We describe here in detail the development of a method to quantitatively measure sphingosine 1-phosphate (Sph-1-P), a bioactive sphingolipid. Sph-1-P was first extracted from cells into the upper aqueous phase under alkaline conditions by Folch's phase separation and then reextracted into the lower chloroform phase under acidic conditions. This phosphorylated sphingoid base extracted was quantitatively converted to N-[3H]-acetylated Sph-1-P, that is [3H]C2-ceramide 1-phosphate (C2-Cer-1-P), by N-acylation with [3H]acetic anhydride. The [3H]C2-Cer-1-P formed with the acylation was resolved by thin-layer chromatography, detected with autoradiography, and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. This assay allows quantification of Sph-1-P over a range from at least 100 pmol (often 30 pmol) to 10 nmol (the highest level tested). The utility and validity of our assay were demonstrated using human platelets. The amount of Sph-1-P in platelet extracts was proportional to the cell number and calculated as 141 +/- 4 pmol/10(8) cells (mean +/- SD, n = 3), which was about four times higher than that of sphingosine. The potent agonist thrombin did not affect the total Sph-1-P amounts in platelet suspensions but induced the release of Sph-1-P stored in the cells into the medium.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vapor-phase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-terminal proline. Since the TFA/water steps have no effect on peptidylthiohydantoins formed from the other 19 amino acids, the additional steps required for proline were readily integrated into the automated sequencing program, providing for the first time an automated sequencing program which permits the C-terminal sequence analysis of all 20 of the commonly occurring amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and larger polypeptides noncovalently applied to Zitex (porous Teflon).

To facilitate investigation of the metabolism of lysophosphatidylcholine and choline lysoplasmalogen in small quantities of tissue, a method for the quantification of these phospholipid species that is capable of accurate and reproducible analysis in samples which contain less than 1 nmol of total choline lysophospholipid was developed. The procedure employs chloroform and methanol extraction of phospholipids from isolated tissue with subsequent separation of the choline lysophospholipid fraction by high-performance liquid chromatography. The choline lysophospholipids are then acetylated with [3H]acetic anhydride and the [3H]acetyl-lysophosphatidylcholine product is isolated by thin-layer chromatography and quantified by liquid scintillation counting. The choline lysophospholipid content in the sample is determined from a standard curve constructed from samples containing a known amount of synthetic lysophosphatidylcholine with correction for recovery based on the inclusion of [14C]lysophosphatidylcholine as an internal standard.

Treatment of glutathione (GSH) with formic acid and acetic anhydride leads to preparation of crystalline N -formyl GSH. This is the first characterized preparation of N -formyl GSH; this product was previously erroneously thought to he N -acetyl GSH. Authentic N -acetyl GSH (crystalline) was prepared by treating GSH with acetic acid and acetic anhydride. The N -acylated derivatives were characterized by 1 H NMR and mass spectroscopy.

A simple and sensitive method for quantification of sphingosine in cellular lipid extracts was developed. The assay is based on quantitative conversion of sphingosine to N-[3H]acetylated sphingosine ([3H]C2-ceramide) by N-acylation with [3H]acetic anhydride under certain conditions. Sphingosine was extracted from cultured cells with chloroform and methanol and then treated with base to remove interfering glycerolipids having reactive amino groups (e.g., phosphatidylethanolamine or phosphatidylserine). Sphingosine was acylated with [3H]acetic anhydride in the presence of 0.004 N NaOH. Acylation was complete in 1 h at 37 degrees C when sphingosine was present in the picomole range. After the acylation, samples were treated with NaOH to reduce background radioactivity by removing the remaining [3H]acetic anhydride and hydrolyzing any ester linkages formed during the acylation and resolved by thin-layer chromatography. [3H]C2-ceramide converted from sphingosine with the acylation was detected with radioautography and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. [3H]C2-ceramide formed was quantitatively measured. This assay allows quantification of sphingosine over a range of 10 to 1500 pmol. The amount of sphingosine in lipid extracts from cultured cells was proportional to the number of cells. Sphingosine levels in human gastric cancer KATO III cells, human promyelocytic leukemic HL60 cells, and human monoblastic U937 cells, determined by this method, were 26.6 +/- 2.2, 6.3 +/- 0.4, and 6.8 +/- 0.6 pmol per 10(6) cells, respectively. Our new procedure allows quantification of sphingosine levels present in the low picomole range in lipid extracts from biological samples.

Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O -bis(trifluoroacetyl)-4-hydroxy- l -proline methyl ester ( N,O -TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N -trifluoroacetyl-4-hydroxy- l -proline methyl ester ( N -TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O -TFA-Hyp ( m z 164, 278) or N -TFA-Hyp ( m z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5–1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18 O 2 incorporation into collagen is possible by selected ion monitoring of the m z 182 ion formed only from the monoacetylated derivative, N -TFA-Hyp, produced by methanol solvolysis of the N,O -TFA-Hyp derivative, as proposed herein.

Variable levels of acetic anhydride have been recommended for addition to one of two reagents used in the glyoxylic acid method for the determination of tryptophan. For use of this reagent immediately after preparation it was shown that a minimum of 16% (v/v) of acetic anhydride should be included in the formulation to obtain near-maximum sensitivity. It was further demonstrated that reagent formulations with and without acetic anhydride changed with exposure to light. The observed changes are manifest as changes in the relative sensitivities of the assay. Several modifications are recommended to improve the sensitivity and stability of the acetic anhydride-containing reagent in this assay.

A gas chromatographic-mass spectrometric method for analysis of l -[2- 15 N]- and l -[5- 15 N]glutamine is described. The method is based on direct acylation of glutamine with trifluoroacetic anhydride and the formation of the N,N -bis-trifluoroacetyl- l -glutamine derivative. This simple and sensitive method is capable of detecting approximately 0.5 atom% excess 15 N in as little as 10 μl of plasma with a mean coefficient of variance of 11.6%. The method was applied to determine the appearance of 15 N enrichment in plasma amino-N and amide-N of glutamine in a healthy adult volunteer during a constant infusion of 15 NH 4 Cl. A plateau level of 3.7 and 2.6 atom% excess was observed in amide-N and amino-N, respectively, at 1 and 2 h after 15 NH 4 Cl infusion was started.

Microgram amounts of proteins applied to polyacrylamide gel electrophoresis were subjected to a fingerprinting procedure using a combined proteolysis-acetylation method with the aid of 14C-labeled acetic anhydride of high specific activity. After staining, gel slices were partially dried and were resoaked in a solution of a protease. After elution and acetylation, the resulting peptides were resolved in fingerprints on cellulose thin-layer chromatography plates and subjected to autoradiography with or without sensitization. Yields, completeness of fingerprinting, and possible artefacts were investigated.

A phenomenological theory of small zone gel chromatography is elaborated for kinetically controlled macromolecule-ligand interactions. Chromatography, direct binding experiments, and rate measurements are used for successful comparison of experimental behavior with theoretical elution profiles for the interaction of MAP-2 with two synthetic peptide fragments from the C-terminal moieties of α- and β-tubulin subunits. The results of this study provide guidelines for interpretation of experimental small zone elution profiles of total ligand.

A method has been developed for attaching peptides to solid supports through their carboxyl groups. The carboxyl groups are activated by mixed anhydride formation in the presence of trifluoroacetic acid and trifluoroacetic anhydride. The activated peptides are then attached to an insoluble resin and subjected to the usual procedures for solid-phase sequencing.

A simplification of the enzymatic isotopic assay for serotonin is described, Serotonin is converted to [ 3 H]melatonin by a two-step reaction: N -acetylation of serotonin using acetic anhydride, followed by O -methylation with the enzyme hydroxyindole O -methyltransferase (EC 2.1,1.4) and S -adenosyl- l -[ methyl - 3 H]methionine as methyl donor. The present assay avoids the use of unstable acetylating enzyme, rat liver N -acetyltransferase (EC2.3,1.5). Blank values are lowered considerably and the sensitivity is doubly increased. Two-tenths micromole of serotonin per 30 μl of sample in tissue homogenates can be measured.