Two-step High-performance Liquid Chromatography Research Articles

6-(2-Formyl-5-methyl-1H-pyrrol-1-yl)hexanoic Acid as a Novel Retronasal Burnt Aroma Compound in Soy Sauce Aroma-Type Chinese Baijiu.

Two-step high-performance liquid chromatography (HPLC) separation in combination with taste dilution analysis was used to screen the retronasal burnt aroma compounds in soy sauce aroma-type Chinese Baijiu (Chinese traditional liquor). A major retronasal burnt aroma region was purified by Sephadex LH-20 column chromatography and HPLC separation from the soy sauce aroma-type Baijiu sample. A novel retronasal burnt aroma compound was identified as 6-(2-formyl-5-methyl-1H-pyrrol-1-yl)hexanoic acid (1) by means of high-resolution mass spectrometry and 1D/2D nuclear magnetic resonance. The retronasal burnt aroma threshold of compound 1 was determined to be 209.5 μg/L in 53% ethanol aqueous solution. Quantitative analysis showed that the concentration of compound 1 ranged from below the limit of quantitation (3.8 μg/L) to 224.2 μg/L in the Baijiu samples. Moreover, the sensory evaluation also revealed that compound 1 may contribute to the retronasal burnt aroma of the Baijiu at its subthreshold concentration by the additive effect with the recently reported retronasal burnt aroma compound 2-hydroxymethyl-3,6-diethyl-5-methylpyrazine.

Analytical and semipreparative chiral separation of cis-itraconazole on cellulose stationary phases by high-performance liquid chromatography.

cis-Itraconazole is a chiral antifungal drug administered as a racemate. The knowledge of properties of individual cis-itraconazole stereoisomers is vital information for medicine and biosciences as different stereoisomers of cis-itraconazole may possess different affinity to certain biological pathways in the human body. For this purpose, either chiral synthesis of enantiomers or chiral separation of racemate can be used. This paper presents a two-step high-performance liquid chromatography approach for the semipreparative isolation of four stereoisomers (two enantiomeric pairs) of itraconazole using polysaccharide stationary phases and volatile organic mobile phases without additives in isocratic mode. The approach used involves the separation of the racemate into three fractions (i.e. two pure stereoisomers and one mixed fraction containing the remaining two stereoisomers) in the first run and consequent separation of the collected mixed fraction in the second one. For this purpose, combination of cellulose tris-(4-methylbenzoate) and cellulose tris-(3,5-dimehylphenylcarbamate) columns with complementary selectivity for cis-itraconazole provided full separation of all four stereoisomers (with purity of each isomer > 97%). The stereoisomers were collected, their optical rotation determined and their identity confirmed based on the results of a previously published study. Pure separated stereoisomers are subjected to further biological studies.

Salivary antigen SP32 is the immunodominant target of the antibody response to Phlebotomus papatasi bites in humans.

BackgroundZoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is highly prevalent in Tunisia and is transmitted by a hematophagous vector Phlebotomus papatasi (P. papatasi). While probing for a blood meal, the sand fly injects saliva into the host's skin, which contains a variety of compounds that are highly immunogenic. We recently showed that the presence of anti-saliva antibodies was associated with an enhanced risk for leishmaniasis and identified the immunodominant salivary protein of Phlebotomus papatasi as a protein of approximately 30 kDa.Methodology/Principal FindingsWe cloned and expressed in mammalian cells two salivary proteins PpSP30 and PpSP32 with predicted molecular weights close to 30 kDa from the Tunisian strain of P. papatasi. The two recombinant salivary proteins were purified by two-step HPLC (High-Performance Liquid Chromatography) and tested if these proteins correspond to the immunodominant antigen of 30 kDa previously shown to be recognized by human sera from endemic areas for ZCL and exposed naturally to P. papatasi bites. While recombinant PpSP30 (rPpSP30) was poorly recognized by human sera from endemic areas for ZCL, rPpSP32 was strongly recognized by the tested sera. The binding of human IgG antibodies to native PpSP32 was inhibited by the addition of rPpSP32. Consistently, experiments in mice showed that PpSP32 induced the highest levels of antibodies compared to other P. papatasi salivary molecules while PpSP30 did not induce any detectable levels of antibodies.ConclusionsOur findings demonstrate that PpSP32 is the immunodominant target of the antibody response to P. papatasi saliva. They also indicate that the recombinant form of PpSP32 is similar to the native one and represents a good candidate for large scale testing of human exposure to P. papatasi bites and perhaps for assessing the risk of contracting the disease.

Purification of antioxidative peptides prepared from enzymatic hydrolysates of tuna dark muscle by-product

Abstract To produce and identify bioactive peptides, two commercial enzymes, orientase (OR) and protease XXIII (PR) were used to hydrolyze tuna dark muscle by-product for up to 6 h, and the hydrolysates were evaluated for antioxidative properties. The results showed that, 60-min OR and 120-min PR hydrolysates possessed the highest antioxidative activity. Then, the protein hydrolysates were subjected to a Sephadex G-25 gel filtration chromatography, and the molecular weight of the peptide fractions which showed the highest antioxidative activity ranged from 390 to 1400 Da. The peptide fractions were further isolated using the two-step high-performance liquid chromatography (HPLC-1 and HPLC-2), and the amino acid sequences of the two antioxidative peptides from OR and PR hydrolysates were Leu–Pro–Thr–Ser–Glu–Ala–Ala–Lys–Tyr (978 Da) and Pro–Met–Asp–Tyr–Met–Val–Thr (756 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna dark muscle by-product may be useful ingredients in food and nutraceutical applications.

Purification of adenoviral vectors by combined anion exchange and gel filtration chromatography

Adenoviral vectors are used extensively in human gene therapy trials and in vaccine development. Large-scale GMP production requires a downstream purification process, and liquid chromatography is emerging as the most powerful mode of purification, enabling the production of vectors at a clinically relevant scale and quality. The present study describes the development of a two-step high-performance liquid chromatography (HPLC) process combining anion exchange (AIEX) and gel filtration (GF) in comparison with the caesium chloride density gradient method. HEK-293 cells were cultured in ten-layer CellStacks() and infected with 10 pfu/cell of adenoviral vector expressing green fluorescent protein (Ad5-GFP). Cell-bound virus was harvested and benzonase added to digest DNA, crude lysate was clarified by centrifugation and filtration prior to HPLC. Chromatography fractions were added to HEK-293 cells and GFP expression measured using a fluorescent plate reader. Using AIEX then GF resulted in an adenoviral vector with purity comparable to Ad5-GFP purified by CsCl, whereas the reverse process (GF-AIEX) showed a reduced purity by electrophoresis and required further buffer exchange of the product. The optimal process (AIEX-GF) resulted in a vector yield of 2.3 x 10(7) pfu/cm(2) of cell culture harvested compared to 3.3 x 10(7) pfu/cm(2) for CsCl. The process recovery for the HPLC process was 36% compared to 27.5% for CsCl and total virion to infectious particle ratios of 18 and 11, respectively, were measured. We present a simple two-step chromatography process that is capable of producing high-quality adenovirus at a titre suitable for scale-up and clinical translation.

Antioxidative properties of peptides prepared from tuna cooking juice hydrolysates with orientase (Bacillus subtilis)

Abstract The antioxidative activity of isolated peptides from of tuna cooking juice hydrolysates by orientase was determined. The results showed that, among the hydrolysates obtained under different hydrolysis time, the highest antioxidative activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity were presented at 60 min. Then, the protein hydrolysate was subjected to a Sephadex G-25 gel filtration chromatography, and five major fractions were obtained. Peptide fractions ranging from 400 to 1500 Da showed the highest antioxidative activity among all fractions. The peptide fractions were further isolated using the two-step high performance liquid chromatography (HPLC-1 and HPLC-2), and six and three fractions were obtained, respectively. The final three antioxidative peptides comprised 4–10 amino acids sequences were observed, and the structures of the peptides were Pro-Val-Ser-His-Asp-His-Ala-Pro-Glu-Tyr (1305 Da), Pro-Ser-Asp-His-Asp-His-Glu (938 Da), and Val-His-Asp-Tyr (584 Da), respectively. We thus conclude that antioxidative hydrolysates from tuna cooking juice may be useful ingredients in food and nutraceutical applications.

Structural classification of acyl‐substituted Quillaja saponins by electrospray ionisation ion trap multiple‐stage mass spectrometry in combination with multivariate analysis

Thirty-eight saponins in two chromatographic fractions (QH-B and QH-C) from Quillaja saponaria Molina have been separated by a two-step high-performance liquid chromatography (HPLC) procedure and investigated by electrospray ionisation ion trap multiple-stage mass spectrometry (ESI-ITMS(n)) in positive ion mode. MS(2) and MS(3) spectra of the compounds were investigated by principal component analysis (PCA) and could be classified by partial least squares - discriminant analysis (PLS-DA) according to the structures of the oligosaccharides at C-3 and C-28 of the saponins. Four minor components with novel structures were found in a previously non-investigated fraction of QH-C. The structures of two of these components, J1 and J1a, were predicted by PLS-DA whereas the structures of the two others, J2 and J3, were only partly predicted. The structures of J1 and J1a were composed of structural elements found in the 34 known saponins whereas a new acyl substituent, not included in the training set used for calibration of the PLS-DA models, was found in J2 and J3, making these two components outliers. The complete structures of the four components were confirmed by monosaccharide analysis, MS(n) data and (1)H NMR spectroscopy.

Colombistatin: a disintegrin isolated from the venom of the South American snake (Bothrops colombiensis) that effectively inhibits platelet aggregation and SK-Mel-28 cell adhesion

Snake venoms are complex mixtures of proteins, which affect the vital biologic systems of prey, as well as humans. Envenomation leads to immobilization by paralysis, cardiac, and circulatory failure. These same venom proteins that cause havoc in the physiologic system could be used as therapeutic agents. Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are non-enzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction. These proteins may have potential in the treatment of strokes, heart attacks, cancers, osteoporosis, and diabetes. The present study describes the isolation and characterization of a disintegrin (colombistatin) found in the venom of the Venezuelan snake mapanare (Bothrops colombiensis). Colombistatin was purified by a two-step high-performance liquid chromatography procedure, which included reverse phase C18 and size exclusion protein Pak 60. Colombistatin inhibited ADP-induced platelet aggregation, human urinary (T24) and skin melanoma (SK-Mel-28) cancer cell adhesion to fibronectin, and cell migration. Colombistatin contained 72 amino acids with a mass of 7.778 kDa as determined by mass spectrometry. Colombistatin could be used as a therapeutic tool in the treatment of melanoma cancers and also thrombotic diseases.

Expression and purification of HtpX-like small heat shock integral membrane protease of an unknown organism related to Methylobacillus flagellatus

The M48 conserved family of peptidases contains a single catalytic zinc ion tetrahedrally co-ordinated by two histidines within an HEXXH motif. The proteases of this class are generally toxic to the cell and thus difficult to express and purify. Here, we report the expression and purification of the small HtpX-like heat shock metalloprotease from an unknown organism related to the obligate methylotrophic anaerobic bacterium, Methylobacillus flagellatus. The protease was expressed in the Escherichia coli vector — pT7. Optimization of expression was done to increase the yield and solubility of the expressed protein. Improved refolding procedures from inclusion bodies of pT7 E. coli system were devised to get the protease in an active and stable form. The protease was purified to near homogeneity in its active form from the refolded proteins of the inclusion bodies by a two-step (cation exchange followed by gel filtration) high performance liquid chromatography (HPLC). The purified protease was active on zymography and casein hydrolysis assays. The activity of the protease was found to be optimum at pH 7.4 and at a temperature of 37 °C but significant activity was also retained at higher temperatures of 45–50 °C. Centrifugal fractionation showed that it is a membrane localized endopeptidase. The methods described here can serve as guidelines to express and purify other homologues of M48 family of proteases for functional and structural studies.

Primary role of growth‐related oncogene‐α and granulocyte chemotactic protein‐2 as neutrophil chemoattractants in chronic rhinosinusitis

The aetiology of chronic rhinosinusitis (CRS) remains unclear. The purpose of this study was to investigate neutrophil-attracting chemokine patterns in CRS without nasal polyposis. The biological activity of the chemokines was identified using a two-step high-performance liquid chromatography (HPLC) technique combined with a bioassay in extracts from 55 CRS patients, and in the turbinate mucosa (TM) of patients (N=51) undergoing septumplasty. The biologic activity of each chemokine was assessed using blocking antibodies to chemokines. Immunolocalization of detected neutrophil chemokines was performed by quantitative evaluation of immunohistochemistry. Besides, PCR analysis was performed to quantify neutrophil chemokine mRNA. In CRS, the chemokines primarily detected by two-step HPLC were growth-related oncogene-alpha (GRO-alpha) and the granulocyte chemotactic protein-2 (GCP-2). Blocking of GCP-2 and GRO-alphad each resulted in chemotaxis inhibition rates of 43.3% and 35.9%, respectively, whereas anti-IL-8 and anti-ENA-78 had no effect. Both GCP-2 and GRO-alphad were generally synthesized by the surface epithelium and mucosal glands while GRO-alpha in particular was synthesized by endothelial cells, as shown by immunohistochemistry. The concentrations of the chemokines IL-8 and epithelial cell-derived neutrophil attractant-78 (ENA-78) were low in CRS and TM. It appears that both GRO-alpha and GCP-2 contribute to neutrophil chemotaxis in CRS, whereas IL-8 and ENA-78 appear to be of secondary importance for the chemotaxis of neutrophils in this condition. The expression of chemokines in mucosal gland cells is the main phenomenon involved in constitutive neutrophil chemotaxis in the TM.

Determination of D- and L-enantiomers of threonine and allo-threonine in mammals using two-step high-performance liquid chromatography

A sensitive and selective method for the determination of four threonine (Thr) isomers (l-Thr, d-Thr, l-allo-Thr and d-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of d-Thr, d-allo-Thr and l-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol–5 pmol per injection, and that of l-Thr is 50 fmol–50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of d-Thr, d-allo-Thr and l-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of d-Thr and d-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of d-Thr (0.85 ± 0.05 nmol/g wet tissue) and d-allo-Thr (5.01 ± 0.32 nmol/g wet tissue) were found in the corpus striatum. l-allo-Thr was not present in any of the tested tissues and physiological fluids.

Determination of d- and l-enantiomers of threonine and allo-threonine in mammals using two-step high-performance liquid chromatography

A sensitive and selective method for the determination of four threonine (Thr) isomers ( l-Thr, d-Thr, l-allo-Thr and d-allo-Thr) in mammalian tissues has been established using two-step high-performance liquid chromatography. This method includes the precolumn fluorescence derivatization of amino acids with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the separation using a combination of a reversed-phase column and a chiral column. The calibration ranges of d-Thr, d-allo-Thr and l-allo-Thr spiked in the rat cerebellum sample are 2.5 fmol–5 pmol per injection, and that of l-Thr is 50 fmol–50 pmol. Within-day and day-to-day precisions of the determination of the four Thr isomers are approximately 5% in the rat cerebellum. By using this method, the tissue distributions of d-Thr, d-allo-Thr and l-allo-Thr in mammals have been demonstrated for the first time in rats, and found that significant amounts of d-Thr and d-allo-Thr are present in the frontal brain areas and urine. Among the 12 tissues tested, the highest amounts of d-Thr (0.85 ± 0.05 nmol/g wet tissue) and d-allo-Thr (5.01 ± 0.32 nmol/g wet tissue) were found in the corpus striatum. l-allo-Thr was not present in any of the tested tissues and physiological fluids.

Synthesis of new carbon nanomolecule: C141

A new [70]fullerene dimer consisting of 141 carbon atoms was first synthesized by a new method with neutron irradiation of [70]fullerene. The results of separation and isolation with two-step HPLC (high performance liquid chromatography) show that this odd-numbered fullerene dimer is stable in air. Laser induced dissociation of C141 mainly yields fragmental species C70 and C71. The UV-vis spectrum of C141 shows that its vis feature is diminished as compared to that of C70. Experiments indicate that this new method for synthesis of fullerene dimers with neutron irradiation has a high selectivity for formation of C2n- X -C2n type carbon nanomolecules.

Purification and Characterization of an Immunomodulatory Endometrial Protein, Glycodelin

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.

Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histones

A new two-step high-performance liquid chromatography (HPLC) procedure has been developed to separate modified histone H1 subtypes. Reversed-phase (RP) HPLC followed by hydrophilic-interaction liquid chromatography (HILIC) was used for analytical and semi-preparative scale fractionation of multi-phosphorylated H1 histone subtypes into their non-phosphorylated and distinct phosphorylated forms. The HILIC system utilizes the weak cation-exchange column PolyCAT A and an increasing sodium perchlorate gradient in a methanephosphonic acid-triethylamine buffer (pH 3.0) in the presence of 70% (v/v) acetonitrile. The identity and purity of the individual histone subfractions obtained was assayed by capillary electrophoretic analysis. The results demonstrate that application of the combined RP-HPLC-HILIC procedure to the analysis and isolation of modified H1 histone subtypes provides an innovative and important alternative to traditional separation techniques that will be extremely useful in studying the biological function of histone phosphorylation.

Plasma levels of trimipramine and metabolites in four patients: determination of the enantiomer concentrations of the hydroxy metabolites.

A two-step high-performance liquid chromatography method is described, using a CN column and an alpha 1-acid glycoprotein column, which allows the measurement of the enantiomers of the hydroxy metabolites of trimipramine in plasma of trimipramine-treated patients. Of the four patients analyzed, three showed approximately equimolar concentrations of the (D)- and (L)-enantiomers of the hydroxy metabolites (2-hydroxy-trimipramine and 2-hydroxy desmethyltrimipramine), and one was found to have roughly twice as much of the (L)-form and of the (D)-form of 2-hydroxy trimipramine and 2-hydroxy desmethyltrimipramine. From the data available on the pharmacological effects of the enantiomers of trimipramine, it is postulated that this interindividual variability in its pharmacokinetics is another factor that could contribute to the interindividual variability in its pharmacodynamics.

High-performance liquid chromatographic separation of hyaluronan and four proteoglycans produced by human bone cell cultures

Four proteoglycans and hyaluronan synthesized by cultured human bone cells were isolated using a two-step high-performance liquid chromatography system involving desalting and buffer exchange with a TSK-GEL HW 40(S) column followed by ion-exchange separation on a Nucleogen 4000-10 DEAE column. The desalting of 4 m guanidinium HCl extracts by a TSK-GEL HW 40(S) column equilibrated in a formamide: KH 2PO 4 buffer produces >95% recoveries, enables quantitation of label incorporation and requires only 40 min to complete. The Nucleogen 4000-10 DEAE column utilizes the same buffer system and requires only 100 min for the resolution of four distinct types of proteoglycans. The formamide:KH 2PO 4 buffer system is compatible with a previously developed polyacrylamide gel system for the electrophoretic profiling of proteoglycans. After separation by charge density, proteoglycans were further resolved by size distribution using a calibrated TSK-GEL HW 75(F) column which also enabled the estimation of the apparent M r of hyaluronan produced by the bone cells. The same TSK-GEL HW 40(S) resin is used to exchange pooled proteoglycans into buffers for analyzing enzyme digests of glycosaminoglycan chains and core proteins. The technique has been applied to the analysis of biosynthetically labeled proteoglycans produced in culture by fetal and adult human bone cells. A distinct pattern of proteoglycan size and secretion for both cell types could be shown using this method. The method of analysis is useful for high yield and rapid screening of various cell types for both biosynthetic rate studies and analysis of patterns of proteoglycan synthesis.

Isolation, characterization and physiological specificity of the crustacean hyperglycemic factors from the sinus gland of the lobster,Homarus americanus(Milne-Edwards)

Summary A two-step high performance liquid chromatography (HPLC) separation system, based on ion exchange and hydrophobic properties, was developed in order to isolate and purify several neuropeptides from the sinus gland of the lobster Homarus americanus. With this system, most of the thus far reported neurohormonal activities, such as hyperglycemia, gonad and pigment controlling factors are detectable. Special attention is given to two pairs of closely related peptides, all reacting with a polyclonal rabbit antiserum raised against the hyperglycemic hormone (CHH) of the crayfish Astacus leptodactylus. Sodium dodecyl sulphate-urea-polyacrylamide gel electrophoresis (SDS-urea-PAGE) of these substances reveals two peptides of about 6.5 kDa, and two more of about 8 kDa. A hyperglycemic response is provoked by both 6.5 kDa peptides and the second 8 kDa component. A stimulatory effect on the oocyte growth is associated with the elution time of both 8 kDa peptides. Furthermore, the presence of two factors with...

Two-step high-performance liquid chromatography method for the determination of myo-inositol and sorbitol

A simple two-step HPLC method for the separation and quantitation of myo-inositol and sorbitol in extracts of glomeruli from rat kidneys is described. The limit of detection is 2 ng. The procedure involves fractionation of the sugar alcohols on a Waters Sugar Pak column, preparation of the p-nitrobenzoate derivatives, and further purification with quantitation by absorbance at 254 nm using a Waters μPorasil column. The applicability of the procedure to determination of sorbitol and myo-inositol in biological samples was demonstrated by the finding of marked alterations in sorbitol and myo-inositol content of glomeruli isolated from diabetic compared to that from normal rat kidneys.

Hydrolysis of 1-alkyl-2-arachidonoyl- sn-glycero-3-phosphocholine, a common precursor of platelet-activating factor and eicosanoids, by human platelet phospholipase A 2

The metabolism of platelet-activating factor (PAF) and arachidonic acid is linked through the common intermediate 1-alkyl-2-arachidonoyl- sn-glycero-3-phosphocholine (alkylarachidonoyl-GPC). Hydrolysis of alkylarachidonoyl-GPC by phospholipase A 2 may initiate the biosynthesis of both PAF and eicosanoids, since alkyllyso-GPC is formed for acetylation to PAF and arachidonic acid is liberated for conversion to biologically active metabolites. In order to elucidate the regulation and functional role of human platelet phospholipase A, in the pathway leading to the formation of both classes of lipid mediators, we have characterized its action upon alkylarachidonoyl-GPC. Human platelet phospholipase A, was solubilized and then partially purified in the presence of n- octyl-β- d- glucopyranoside (octyl glucoside). Hexadecylarachi-donoyl-GPC was prepared biosynthetically using platelet sonicates, purified by two-step high-performance liquid chromatography (HPLC) and suspended in buffer by sonication. Our results indicate that deacylation of alkylarachidonoyl-GPC by platelet phospholipase A 2 has an absolute requirement for Ca 2+. It occurs at submicromolar concentrations of free Ca 2+ and exhibits a biphasic Ca 2+-dependence with activity plateaus at 10 μM and 2 mM. Phospholipase A 2-mediated hydrolysis of alkylarachidonoyl-GPC is increased 2-fold by albumin and is enhanced 5-fold if 1,2-dioleoylglycerol is incorporated into the substrate dispersion. The substrate dependence and specificity of platelet phospholipase A 2 for 1-alkyl- vs. 1-acyl-linked subclasses of arachidonic acid containing phosphatidylcholine was examined with 1- O-hexadecyl-2-arachidonoyl- sn-glycero-3-phosphocholine (hexadecylarachidonoyl-GPC) and l-palmitoyl-2-arachidonyl- sn-glycero-3-phosphocholine (palmitoylarachidonoyl-GPC). We found that the substrates were deacylated equivalently. We conclude that, in stimulated platelets, in the presence of increased levels of cytoplasmic free Ca 2+ and newly generated diacylglycerol, alkylarachidonoyl-GPC may be rapidly hydrolyzed by phospholipase A 2 and may serve as a precursor of both PAF and eicosanoids.