Content Of Hydrophobic Amino Acids Research Articles

Effect of Ultrasound on Alkali Extraction Protein from Rice Dreg Flour

AbstractIn this study, Counter‐current ultrasound‐assisted alkali was employed to extract rice dreg protein (RDP) from rice dreg flour (RDF). The results indicated that extraction yield of RDP reached a maximum value at 0.08 mol/L alkali concentrate. It was only 43.20% in absence of ultrasound, whereas it reached up to 88.44% under ultrasonic condition. Amino acid composition, scanning electron microscopy, atomic force microscopy and fourier transform infrared spectra were used to determine amino acid composition, morphology, aggregates and microstructure of RDP. Ultrasound treatment could result in improving of hydrophobic amino acids content and changing of RDF and RDP microstructure, showing enhancement in the porosity of RDF particle, variation of RDP particle size. Thus, the acceleration of protein extraction was related to the changes of RDF and RDP microstructure caused by ultrasound. Counter‐current ultrasound‐assisted alkali extraction, as a modified method, was proved to be helpful in industrial manufacture of RDP from RDF.Practical ApplicationsLow‐frequency high‐intensity ultrasound has been used in many aspects in laboratory, such as extraction of components, pretreatment. Counter‐current ultrasound‐assisted extraction was proved to be helpful in industrial manufacture of protein because it can dramatically improve extraction rate of rice dreg protein, and Counter‐current ultrasound method can achieve continuous production of industrialization.

Solid-state fermentation of Acanthogobius hastaprocessing by-products for the production of antioxidant protein hydrolysates with Aspergillus oryzae

Functional properties and antioxidative activity of a protein hydrolysate prepared from Acanthogobius hasta processing by-product protein during solid-state fermentation with Aspergillus oryzae were investigated. Overall, protease activity increased with the degree of hydrolysis (DH) decreased during solid-state fermentation. All the protein hydrolysate had excellent solubility, possessed interfacial properties, and varying degrees of antioxidant activity which were governed by their concentrations and DH, molecular weight distribution and amino acid composition. After 5 days fermentation, the DH of the protein hydrolysate was 31.23%. The protein hydrolysate had the highest total hydrophobic amino acid content, the highest DPPH scavenging activity, reducing power, and the chelating activity. The radical-scavenging activity of the hydrolysates at 6 mg/mL was 78.6%. The reducing power of protein hydrolysate at the range of 0-6 mg/mL was lower than that of BHA at the range of 0-60 µg/mL, while the chelating activity of APs was similar to that of BHA at the range of 0-60 µg/mL. Moreover, the protein hydrolysate showed good emulsifying and foaming properties over a wide pH range from 2 to 12. Therefore, solid state fermentation provided a suitable and low-cost method for converting Acanthogobius hasta processing by-product protein into antioxidant protein hydrolysates.

Isolation and identification of a high molecular weight protein in sow milk

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.

Bp-13 PLA2: Purification and Neuromuscular Activity of a New Asp49 Toxin Isolated from Bothrops pauloensis Snake Venom.

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°–45°C. Full Bp-13 PLA2 activity required Ca2+; its PLA2 activity was inhibited by Mg2+, Mn2+, Sr2+, and Cd2+ in the presence and absence of 1 mM Ca2+. In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca2+ by Sr2+ and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.

Influence of Sorghum Kafirin on Serum Lipid Profile and Antioxidant Activity in Hyperlipidemic Rats (In Vitro and In Vivo Studies).

The aim of this study was to compare in vitro the antioxidant potential of sorghum kafirin and sorghum flour and their influence on lipids and antioxidant capacity in rats. The antioxidant activity in sorghum kafirin extract measured by the DPPH and TEAC methods was increased 30 and 65 times, respectively, compared to that of its counterpart, sorghum flour. According to electrophoresis assay, the kafirins tert-butanol extract showed a high proportion of α-kafirin monomers, and its amino acid composition revealed higher hydrophobic amino acid content such as alanine, isoleucine, leucine, tyrosine and phenylalanine than sorghum flour extract. Diets supplemented with sorghum kafirin extract have improved lipid metabolism and increased the serum antioxidant potential (67%) especially in rats fed with added cholesterol. The bioactive peptides generated from kafirin in vivo hydrolysis appear to be associated with the positive effect on serum lipids and antioxidant activity. According to these results, sorghum kafirin extract at the levels used in this study apparently could be used for prevention of atherosclerosis and other chronic diseases.

ACE Inhibitory and Antioxidant Activities of Collagen Hydrolysates from the Ribbon Jellyfish (Chrysaora sp.).

Collagen isolated from the ribbon jellyfish (Chrysaora sp.) was hydrolysed using three different proteases (i.e. trypsin, alcalase and Protamex) to obtain bioactive peptides. Angiotensin-I-converting enzyme (ACE) inhibitory activity and antioxidant activities (i.e. ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity) of the peptides were measured and compared, and the effect of the duration of hydrolysis on the bioactivity (ACE inhibitory and antioxidant activities) of peptides was also evaluated. FRAP activity was the highest in Protamex-induced (25-27 mM) and trypsin-induced hydrolysates (24-26 mM) at 7 and 9 h, respectively. Conversely, hydrolysates produced by trypsin for 1 and 3 h showed the highest DPPH radical scavenging activities (94 and 92%, respectively). Trypsin-induced hydrolysates (at 3 h) also showed the highest ACE inhibitory activity (89%). The peptide sequences with the highest activities were identified using tandem mass spectrometry, and the results show that the hydrolysates had a high content of hydrophobic amino acids as well as unique amino acid sequences, which likely contribute to their biological activities.

Antibacterial activity of new peptides from barbel protein hydrolysates and mode of action via a membrane damage mechanism against Listeria monocytogenes

There is great interest in the development of antimicrobial peptides as a potentially novel class of antimicrobial agents. In this study we evaluated the mode of action of new peptides (Gly-Val-His, Trp-His-Arg, Trp-His-Phe, Pro-Pro-Ser-Ser, Ala-Ala-Ala-Leu, Ala-Ala-Gly-Gly-Val, Ala-Ala-Val-Lys-Met, Ala-Ser-Ser-Ser), previously characterized, from barbel protein hydrolysates against Listeria monocytogenes via a membrane damage mechanism. The minimum inhibitory concentrations (MIC) of these peptides toward six strains and their hemolytic activity toward bovine erythrocytes were determined.Prediction of peptide secondary structure indicated that these peptides should have random coil structures and high content of hydrophobic amino acids. In addition, results of the determination of extracellular potassium, which is considered a good marker of membrane permeability, revealed that treatment with pure barbel peptides could cause morphological changes of L. monocytogenes and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from fish protein hydrolysates.

Antioxidant activities of rice bran protein hydrolysates in bulk oil and oil-in-water emulsion.

Recently, utilization of natural antioxidants in food processing has been of growing interest, owing to the concerns of health hazards of synthetic agents. Protein hydrolysates are a potent candidate for this purpose. In this work, rice bran protein hydrolysates (RBPH) with various degrees of hydrolysis (DH) were prepared, and their antioxidant activities in soybean oil and oil-in-water (O/W) emulsion were examined. With increasing DH, RBPH showed increasing antioxidant activities, as evidenced by the increases in DPPH radical scavenging activity, reducing power and ferrous chelating activity (P < 0.05). The improved activity was associated with increasing surface hydrophobicity (SoANS). After hydrolysis for 60 min, the content of hydrophobic amino acids was increased. When RBPH with various DH were incorporated into bulk soybean oil and O/W emulsion stored at 37 °C for up to 15 days, lipid oxidation was successfully retarded, especially when DH increased. The efficiency in prevention of oxidation was dose dependent (0-10 g L(-1)), as indicated by the lower peroxide value and thiobarbituric reactive substances. The present work suggests that RBPH might be potently employed as a natural antioxidant in both bulk oil and emulsion models.

Optimization and biochemical characteristics of an enzymatic squid hydrolysate for manufacture of a squid complex seasoning

Enzymatic hydrolysis of squid meat was optimized and the biochemical properties of solubility, amino acid composition, and contents of nucleic acid related compounds were determined. Optimal hydrolysis conditions were a pH of 7.0, a hydrolysis time of 5.9 h, a temperature of 55°C, and an enzyme/substrate ratio of 2.4%. More than 75% of squid meat hydrolysate was soluble in a wide pH range. The sweet and savory tasting amino acid contents of the squid hydrolysate were higher than the contents of bitter tasting amino acids. The hydrophilic amino acid content of squid meat was lower than the content of hydrophobic amino acids. The inosine monophosphate content of squid hydrolysate was highest among nucleic acid related compounds. Based on a sensory evaluation, squid complex seasoning was similar or superior to commercial complex seasonings. A squid hydrolysate can be used as an ingredient for manufacture of food products.

Comparison of physicochemical properties, antioxidant and metal‐chelating activities of protein hydrolysates from Phaseolus lunatus and hard‐to‐cook Phaseolus vulgaris

SummaryLimited and extensive hydrolysates were obtained from Phaseolus lunatus (LHl and EHl) and hard‐to‐cook Phaseolus vulgaris (LHv and EHv) using the enzymes Flavourzyme®, Alcalase®, Pancreatin® and a sequential Pepsin®–Pancreatin® system. Degrees of hydrolysis varied from 8.32% to 31.60%. SDS‐PAGE of extensive hydrolysates showed molecular weights smaller than limited hydrolysates. Differential scanning calorimeter (DSC) analysis of LHl and EHl revealed the presence of two endothermic transitions; LHv and EHv had only one. LHv presented a higher content of hydrophobic amino acids whose surface hydrophobicity was 12.17. Functional properties such as nitrogen solubility, foaming capacity and emulsifying activity index in LHv were better than LHl at different pH evaluated. However, the latter showed better foaming stabilities. Amino acids such as His, Tyr, Trp and Arg were observed in greater amounts in both extensive hydrolysates. 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical‐scavenging and metal‐chelating activities in EHv and EHl increased significantly compared to the source material.

Evaluation of Antimicrobial, Antioxidant Activities, and Nutritional Values of Fermented Foxtail Millet Extracts by Lactobacillus paracasei Fn032

Solid-state bioprocessing of foxtail millet by Lactobacillus paracasei Fn032 is a biotechnological strategy to produce fermented foxtail millet meal with more beneficial components. The objective of this study was to evaluate the antioxidant, antimicrobial properties and nutritional values of water extracts from fermented foxtail millet flour and its bran with and without protease. Fermented foxtail millet flour with added protease extract showed higher scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals as well as reducing power than fermented foxtail millet flour and fermented foxtail millet bran extracts. Both extracts, fermented foxtail millet flour and fermented foxtail millet flour with protease, showed significant (P < 0.05) effectiveness inhibition abilities on microbial growth when compared with fermented foxtail millet bran extracts. Amino acid profile revealed that fermented foxtail millet flour with protease, with relatively strongest antioxidant, antimicrobial activity, also had the highest total hydrophobic amino acids content (51.39%) and hydrophobic index (8.47 Kj/mol amino acid residue). Moreover, fermented foxtail millet flour with protease revealed the highest protein content, predicted protein efficiency ratio, and protein digestibility. Molecular weight of the whole extracts varied from 180–5000 Da. Based on the results obtained, fermented foxtail millet flour extracts were relatively effective in the antioxidant, antimicrobial properties assayed and might be potential biological values for application in food products.

Calcium-Binding Capacity of Wheat Germ Protein Hydrolysate and Characterization of Peptide–Calcium Complex

This study investigates the ability of various wheat germ protein hydrolysates (WGPHs) to bind calcium and characterizes the peptide-calcium complexes. We demonstrate that the amount of Ca bound depended greatly on the type of enzyme, degree of hydrolysis (DH), amino acid composition, and molecular mass distribution of different hydrolysates. The maximum level of Ca bound (67.5 mg·g(-1)) occurred when Alcalase was used to hydrolyze wheat germ protein at a DH of 21.5%. Peptide fragments exhibiting high calcium-binding capacity had molecular mass <2000 Da. The calcium-binding peptides mainly consisted of Glu, Arg, Asp, and Gly, and the level of Ca bound was related to the hydrophobic amino acid content in WGPHs. UV-visible and Fourier transform infrared spectra demonstrate that amino nitrogen atoms and oxygen atoms on the carboxyl group were involved in complexation. Therefore, wheat germ protein is a promising protein source for the production of calcium-binding peptides and could be utilized as a bioactive ingredient for nutraceutical food production.

Improving the antibacterial activity and selectivity of an ultra short peptide by hydrophobic and hydrophilic amino acid stretches

The principle of amino acid stretches tagged at the C terminal of Luecrocin I, which is an ultra-short antibacterial peptide, by tryptophan and arginine or lysine has been reported. The choice of amino acid type at each stretch position depends on the hydrophobic and hydrophilic regions visualized in the helical wheel pattern of Luecrocin I. Oligopeptide tagging should also consider the properties such as positive charge, hydrophobicity, the content of hydrophobic amino acids, polar angle, the properly hydrophilic and hydrophobic facets. Amidation at C terminal and lysine substitute for arginine can increase selectivity between mammalian cells (hemolytic and MTT assay) and bacterial cells tested. KT2 and RT2 which have 53% hydrophobic residues, 7 positive charges, 160° polar angle, −0.02 (KT2) and −0.04 (RT2) hydrophobicity were effective against S. typhi DMST 22842, S. epidermidis ATCC 12228, E. coli ATCC 25922 and V. cholerae non-O1, non-O139. The SEM images implied that the antibacterial mechanism of RT2 and KT2 may depend on concentration rather than time. Finally, RT2 and KT2 can be new antibacterial agents or may be further developed for alternative antibiotics.

Reverse-phase HPLC Separation of Hemp Seed (Cannabis sativa L.) Protein Hydrolysate Produced Peptide Fractions with Enhanced Antioxidant Capacity

Hemp seed protein hydrolysate (HPH) was produced through simulated gastrointestinal tract (GIT) digestion of hemp seed protein isolate followed by partial purification and separation into eight peptide fractions by reverse-phase (RP)-HPLC. The peptide fractions exhibited higher oxygen radical absorbance capacity as well as scavenging of 2,2-diphenyl-1-picrylhydrazyl, superoxide and hydroxyl radicals when compared to HPH. Radical scavenging activities of the fractionated peptides increased as content of hydrophobic amino acids or elution time was increased, with the exception of hydroxyl radical scavenging that showed decreased trend. Glutathione (GSH), HPH and the RP-HPLC peptide fractions possessed low ferric ion reducing ability but all had strong (>60%) metal chelating activities. Inhibition of linoleic acid oxidation by some of the HPH peptide fractions was higher at 1mg/ml when compared to that observed at 0.1mg/ml peptide concentration. Peptide separation resulted in higher concentration of some hydrophobic amino acids (especially proline, leucine and isoleucine) in the fractions (mainly F5 and F8) when compared to HPH. The elution time-dependent increased concentrations of the hydrophobic amino acids coupled with decreased levels of positively charged amino acids may have been responsible for the significantly higher (p < 0.05) antioxidant properties observed for some of the peptide fractions when compared to the unfractionated HPH. In conclusion, the antioxidant activity of HPH after simulated GIT digestion is mainly influenced by the amino acid composition of some of its peptides.

Biochemical Characterization and Pharmacological Properties of New Basic PLA2BrTX-I Isolated fromBothrops roedingeri(Roedinger's Lancehead) Mertens, 1942, Snake Venom

BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on μ-Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35–45°C, and required Ca2+. In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNFα were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively.

Renin and angiotensin converting enzyme inhibition with antioxidant properties of African yam bean protein hydrolysate and reverse-phase HPLC-separated peptide fractions

Enzymatic hydrolysate of African yam bean seed proteins was prepared by treatment with alcalase. The African yam bean seed protein hydrolysate (APH) was further separated into six fractions (F1–F6) on a C12 reverse-phase HPLC column. With exception of F6, amino acid composition analysis showed increase in content of hydrophobic amino acids as residence time on the column increased. Some of the peptide fractions had significantly higher (p<0.05) superoxide and DPPH radical scavenging activities as well as higher inhibition of linoleic acid oxidation when compared to the unfractionated APH. Iron-chelating ability of APH and peptide fractions was positively related to inhibition of linoleic acid oxidation, which suggests the role of metal-catalyzed reactions during lipid peroxidation. However, peptide fractionation had no effect on ferric reducing antioxidant power. F4 and F6 fractions had significantly higher ACE- and renin-inhibitory properties, respectively. The results showed that a higher residency time on the column (reflecting stronger net hydrophobic character) enhanced peptide-induced renin inhibition but lower net hydrophobic character was better for ACE inhibition. There was no direct relationship between ACE and renin inhibitions, indicating different modes of enzyme-catalyzed reactions. We concluded that the APH and peptide fractions may have potential uses as antioxidants and modulators of the renin–angiotensin system for the management of oxidative stress and cardiovascular disorders, respectively.

Alpha‐Helix Self‐Assembly of Oligopeptides Originated From Beta‐Sheet Keratin

AbstractThe structure and characterization of the oligopeptide crystals formed from the feather keratin solution obtained by superheated water treatment are reported. The FTIR spectra and 1H and 13C solid‐state NMR results indicate that the peptides, arranged mostly in a beta‐sheet structure in feather, reorganize into a mainly alpha‐helix and less beta‐sheet mixed secondary structure, when self‐assemble from the solution at room temperature. MALDI‐ToF‐ToF spectra confirm that the most primary sequence with the mass 1884 come from the feather keratin 4, KRFA_CHICK of Gallus gallus. The largely preservation of all but cystine amino acid species and the increase of hydrophobic amino acids content in the oligopeptide crystals are proved by the amino acid analysis.

ACE inhibitory activity of pangasius catfish (Pangasius sutchi) skin and bone gelatin hydrolysate.

Skin and bone gelatins of pangasius catfish (Pangasius sutchi) were hydrolyzed with alcalase to isolate Angiotensin Converting Enzyme (ACE) inhibitory peptides. Samples with the highest degree of hydrolysis (DH) were separated into different fractions with molecular weight cut-off (MWCO) sizes of 10, 3 and 1kDa, respectively and assayed for ACE inhibitory activity. Skin and bone gelatins had highest DH of 64.87 and 68.48% after 2 and 1h incubation, respectively. Results from this study indicated that by decreasing the molecular weight of fractions, ACE inhibitory activity was increased. Therefore, F3 permeates (MWCO < 1kDa) of skin (IC50 = 3.2μg/ml) and bone (IC50 = 1.3μg/ml) gelatins possessed higher ACE inhibitory activity compared to their untreated gelatins and corresponding hydrolyzed fractions. In this study, the major amino acids were Glycine followed by Proline with an increased amount of hydrophobic amino acid content in F3 permeates of skin (4.01%) and bone (5.79%) gelatin. Digestion stability against gastrointestinal proteases did not show any remarkable change on ACE inhibition potency of these permeates. It was concluded that alcalase hydrolysis of P. sutchi by-products could be utilized as a part of functional food or ingredients of a formulated drug in order to control high blood pressure.

Investigation of ORF0 as a sensitive alternative diagnostic segment to detect Sugarcane yellow leaf virus

The worldwide distribution of Sugarcane yellow leaf virus (SCYLV) has led several research groups to study the function of the viral genome, the role of open reading frames (ORFs), their influence on virus accumulation and methods for diagnosis. The detection of SCYLV is usually based on the viral coat protein whether using serological and/or molecular techniques. In this study, ORF0 has been used as a diagnostic segment for SCYLV due to its highly conserved region in all SCYLV isolates. The results revealed that, ORF0 was expressed more consistently in all cultivars. In contrast, the expression of the coat protein varied. The RNA poleroviruses sequences of ORF0 were variable compared with the same segment of SCYLV populations. Analysis of the amino acid sequence of the ORF0 translation product revealed the presence of a potential transmembrane domain. The relatively high content of hydrophobic amino acids in the ORF0 protein further suggests that it may serve as a membrane anchor for the replication complex.

Enzymatic measurement of phosphatidylserine in cultured cells

Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid involved in diverse cellular functions. In this study, we developed a new fluorometric method for measuring PS using combinations of specific enzymes and Amplex Red. The calibration curve for PS measurement was linear and hyperbolic at low (0-50 µM) and high (50-1000 µM) concentrations, respectively, and the detection limit was 5 µM (50 pmol in the reaction mixture). This assay quantified PS regardless of the chain length and the number of double bonds. We applied this new method to the determination of PS content in HEK293 cells, which was validated by a recovery study and comparison with TLC-phosphorus assay. We showed that the PS content was high in sparse cells. The overexpression of PS synthase 1 elevated not only the cellular PS content but also the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents, suggesting the conversion of PS into PE and the enhancement of PC production. This new assay for PS measurement is simple, specific, sensitive, and high throughput, and it will be useful to clarify the metabolism and biological functions of PS.