Acylated Forms Research Articles

Decreasing pH results in a reduction of anthocyanin coprecipitation during cold stabilization of purple grape juice.

Anthocyanin pigments in grape juice can coprecipitate with potassium bitartrate (KHT) crystals during cold stabilization, but factors that reduce these adsorptive losses are not well understood. We hypothesized that coprecipitation on a % w/w basis should be decreased at lower pH. In initial experiments, model juice solutions containing an anthocyanin monoglucoside extract and varying pH values were subjected to cold-storage to induce KHT crystallization, and anthocyanins in the resulting precipitant were characterized by HPLC. The pH of the model juice was directly correlated with the % w/w concentration of anthocyanins in the KHT crystals, with a maximum observed at pH 3.40 (0.20% w/w) and a minimum at pH 2.35 (0.01% w/w). A pH dependency was also observed for anthocyanin-KHT coprecipitation in purple Concord grape juice, although the effect was smaller. Coprecipitation was significantly greater for anthocyanin monoglucosides and acylated anthocyanins as compared to anthocyanin diglucosides at pH > 3.05, but coprecipitation of mono- and acylated forms declined more sharply at lower pH values.

Screening reactivity of molecules 6-aminopenicillanic acid and its acylated forms according to the calculation results molecular modeling

Screening reactivity of molecules 6-aminopenicillanic acid and its acylated forms according to the calculation results molecular modeling

Stability of anthocyanins from red grape skins under pressurized liquid extraction and ultrasound-assisted extraction conditions.

The stability of anthocyanins from grape skins after applying different extraction techniques has been determined. The following compounds, previously extracted from real samples, were assessed: delphinidin 3-glucoside, cyanidin 3-glucoside, petunidin 3-glucoside, peonidin 3-glucoside, malvidin 3-glucoside, peonidin 3-acetylglucoside, malvidin 3-acetylglucoside, malvidin 3-caffeoylglucoside, petunidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside (trans). The techniques used were ultrasound-assisted extraction and pressurized liquid extraction. In ultrasound-assisted extraction, temperatures up to 75 °C can be applied without degradation of the aforementioned compounds. In pressurized liquid extraction the anthocyanins were found to be stable up to 100 °C. The relative stabilities of both the glycosidic and acylated forms were evaluated. Acylated derivatives were more stable than non-acylated forms. The differences between the two groups of compounds became more marked on working at higher temperatures and on using extraction techniques with higher levels of oxygen in the extraction media.

Erratum to: Biosynthesis of phenolic compounds in Vitis vinifera cell suspension cultures: Study on Hydroxycinnamoyl CoA: ligase

In cell suspensions cultures from grape berry pulp (Vitis vinifera cv. Gamay fréaux)hydroxycinnamoyl CoA ligase (CoAL) displayed maximum activity (100 %) forp-coumaric acid and then, in decreasing order, for ferulic acid (81.3 %) and caffeic acid (60.4 %). No activity was detected with sinapic and cinnamic acids. The changes in CoAL activity during the growth cycle of the culture displayed two peaks : the highest (6 h after subculturing) was linked with a strong increase in protein caused by dilution ; the second was weaker and occurred on the 7th day of culture.Grape cell suspension accumulated mainly peonidin (Pn) and cyanidin (Cy) glucosides (Pn 3-glucoside, Cy 3-glucoside, Pn 3-acetylglucoside, Pn 3-caffeylglucoside, Pn 3-p-coumarylglucoside, and Cy 3-p-coumarylglucoside). Maximum accumulation of anthocyanins was associated with the exponential growth phase of the culture and might be the result of the substantial increase in CoAL activity resulting from the effect of dilution. The second enzyme activity peak was probably oriented towards the acylation of anthocyanins since the percentage of acylated forms increased with time after subculturing.

РіДИННОФАЗНЕ КАТАЛіТИЧНЕ ОКИСНЕННЯ ТОЛУОЛУ ОЗОНОПОВіТРЯНОЮ СУМіШШЮ

The article presents the results of the study of kinetic patterns of the reaction of ozone with toluene in acetic anhydride. It was shown that an effective method of control of the depth of ozone oxidation of toluene at the methyl group is carrying out the reaction in a solution of acetic anhydride with sulfuric acid, where the labile intermediates in the moment of formation turn into the forms resistant to ozone - benzyl acetate and benzylidene diacetate. The use of manganese-bromide catalyst increases the rate and depth of oxidation of toluene: the main products of the reaction are benzaldehyde (72.5% in acylated and 10% in non acylated forms and 7.5% of benzyl acetate. It was determined that the implication of toluene to the oxidation at the methyl group passes according to the reaction with the oxidized form of the metal, which is formed in result of the reaction with ozone. A scheme for catalysis was suggested in the article. In these conditions, the oxidation is carried out by molecular oxygen, and ozone acts as an initiator of oxidation. The kinetics of the main stages of the catalytic cycle was studied, the mechanism of oxidation-reduction catalysis, which conforms to the experimental data obtained was suggested

Binding of polyphenols to plant cell wall analogues – Part 1: Anthocyanins

Anthocyanins are located within the vacuole of plant cells, and are released following cell rupture during eating or processing at which time they first come into contact with the plant cell wall. The extent of anthocyanin-cell wall interaction was investigated by monitoring the rate of anthocyanin depletion in the presence of pure cellulose or cellulose-pectin composites as cell wall models. It was found that anthocyanins interact with both cellulose and pectin over a two-stage process with initially (mins-hours) 13 similar to 18% of anthocyanins binding to cellulose or cellulose/pectincomposites. With prolonged exposure (days-weeks), a gradual increase in anthocyanin binding occurs, possibly due to anthocyanins stacking on top of a base layer. Binding of acylated and non-acylated anthocyanins followed a similar pattern with slightly more (5-10%) binding of the acylated forms. Composites with the highest pectin content had the greatest anthocyanin binding suggesting the existence of both ionic interactions (with pectin) and hydrophobic interactions (with cellulose) of anthocyanin with plant cell walls.

Bunch exposure to direct solar radiation increases ortho-diphenol anthocyanins in Northern Italy climatic condition

<p style="text-align: justify;"><strong>Aims</strong>: Berry anthocyanin contents were studied in eight different field experiments involving various grapevine (<em>Vitis vinifera</em> L.) cultivars (Syrah, Croatina, Sangiovese, Nebbiolo, Nebbiolo Rosé, Carmènere and Cannonau) in a variety of sites located in Northern Italy.</p><p style="text-align: justify;"><strong>Methods and results</strong>: In each experiment, global irradiance was reduced by approximately 95% by applying specially designed boxes to selected bunches before the onset of ripening. Variations in anthocyanin accumulation and profile were studied on sun exposed and shaded bunches. Exposed bunches were subjected to less favorable temperatures for biosynthetic metabolism than shaded bunches, due to frequent excessive temperatures around noon.</p><p style="text-align: justify;"><strong>Conclusions</strong>: Exposure to direct solar radiation induced an earlier and faster anthocyanin accumulation and a higher anthocyanin concentration across all the experiments, at least from veraison to mid ripening. In fact, the light induced elevated rate of anthocyanin accumulation did not always result in a higher anthocyanin content at grape maturity. In the cultivars with a relatively high proportion of acylated forms, sun exposure resulted in a relative increase in the quantities of non-acylated glucoside forms, while the acylation profile of cultivars that generally had a lower proportion of acylated forms was only minimally influenced by sun exposure.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Akey observation resulting from anthocyanin profiling is the strong positive correlation between light exposure and increases in the percentage of delphinidin-, cyanidin- and petunidin-3-monoglucoside, that is, anthocyanins containing an orthodihydroxyl group.</p>

Identification of the Phenolic Components of Collard Greens, Kale, and Chinese Broccoli

An LC-MS profiling method was used for a comprehensive study of the phenolic components of collard greens, kale, and Chinese broccoli, three Brassica green leafy vegetables. This study led to the identification of 45 flavonoids and 13 hydroxycinnamic acid derivatives in the three vegetables. Most of the identifications were based on comparison of compounds previously reported in the literature for Brassica vegetables. The results indicate that the three materials have very similar phenolic component profiles. For each, kaempferol glycosides and acylgentiobiosides were the major phenolic compounds, quercetin glycosides were minor compounds, and most of the flavononol glycosides existed in their acylated forms. In addition, each of the materials contained caffeoyl-, p-coumaroyl-, and feruloylquinic acid monomers with a 3-position derivative as the dominant isomer. This is the first report for most of these phenolics in collard greens and Chinese broccoli and for >20 of them in kale.

Probing the Interactions of Early Polyketide Intermediates with the Actinorhodin ACP from S. coelicolor A3(2)

Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. Seven thiol ester and thiol ether derivatives of the actinorhodin (act) PKS ACP from Streptomyces coelicolor have been prepared and structurally characterised by NMR to gain insight into ACP-intermediate interactions. Holo ACP synthase has been used to prepare early-stage ACP intermediates of polyketide biosynthesis (holo ACP, acetyl ACP, and malonyl ACP) from the respective coenzyme A derivatives. A synthetic route to stabilised thiol ether ACPs was developed and applied to the preparation of stable 3-oxobutyl and 3,5-dioxohexyl ACP as diketide and triketide analogues. No interaction between the protein and the acyl phosphopantetheine moieties of acetyl, malonyl, or 3-oxobutyl ACP was detected. Analysis of (1)H-(15)N heteronuclear single quantum coherence and nuclear Overhauser enhancement spectroscopy spectra for the triketide ACP revealed exchange between a major ('Tri', 85%) and a minor protein conformer in which the polyketide interacts with the protein ('Tri(*)', 15%). Act ACP was also derivatised with butyryl, hexanoyl, and octanoyl groups. The corresponding NMR spectra showed large chemical shift perturbations centred on helices II and III, indicative of acyl chain binding and significant structural rearrangement. Unexpectedly, butyryl act ACP showed almost identical backbone (1)H-(15)N chemical shifts to Tri(*), suggesting comparable structural changes that might provide insight into the structurally uncharacterised polyketide bound form. Furthermore, butyryl ACP itself underwent slow conformational exchange with a second minor conformer (But(*)) with almost identical backbone chemical shifts to octanoyl act ACP. High-resolution NMR structures of these acylated forms revealed that act ACP was able to undergo dramatic conformational changes that exceed those seen in FAS ACPs. When compared to E. coli FAS ACP, the substrate binding pocket of the act PKS ACP has three specific amino acid substitutions (Thr39/Leu45, Ala68/Leu74, and Leu42/Thr48) that alter the size, shape, and location of this cavity. These conformational changes may play a role in protein-protein recognition and assist the binding of bulky polyketide intermediates.

Stabilization of α-chymotrypsin in aqueous organic solvents by chemical modification with organic acid anhydrides

When the primary amino groups of α-chymotrypsin were modified with acetic, propionic, succinic, citraconic and phthalic acid anhydrides, the modifications enhanced the stability of the enzyme in 60% aqueous solutions of 1,4-dioxane, ethanol and acetonitrile. The acetylation of the amino groups resulted in the lowest stabilization, but the citraconylated, propionylated and succinylated forms of α-chymotrypsin exhibited increased stabilities in all the aqueous organic solvents studied. The modification of α-chymotrypsin with phthalic anhydride was accompanied by very extensive activation. The near-UV circular dichroism spectroscopic measurements did not indicate significant structural changes in the acylated forms of α-chymotrypsin, with the exception of the phthalic anhydride-modified enzyme. The spectral changes in the phthalic anhydride-modified enzyme suggest enhanced interactions between the aromatic chromophores and alterations in the disulfide contribution. The improvement in stability may be related to the modifications caused by the reorientation and the increased interactions of the aromatic side-chains, particularly in the case of the phthalic anhydride derivative.

Berry Temperature and Solar Radiation Alter Acylation, Proportion, and Concentration of Anthocyanin in Merlot Grapes

Using a forced convection system, temperatures of Merlot grape clusters were monitored and controlled between veraison and harvest to produce a dynamic range of berry temperatures under field conditions in both sun-exposed and shaded fruit. Ten combinations of temperature and solar radiation exposure were used to quantify effects on phenolic profiles (anthocyanins and flavonol-glycosides) and on total concentrations of skin anthocyanin (TSA) in the fruit at commercial maturity. Exposure of berries to high temperature extremes for relatively short periods during ripening appears to alter the partitioning of anthocyanins between acylated and nonacylated forms and between dihydroxylated and trihydroxylated branches of the anthocyanin biosynthetic pathway. Specifically, among flavonol-glycosides, quercetin 3-glucoside increased with exposure to solar radiation. Low incident solar radiation alone appeared not to compromise total anthocyanin accumulation; rather, a combination of low light and high berry temperatures decreased TSA. Regardless of exposure to solar radiation, higher berry temperatures led to a higher concentration and a higher proportion of TSA comprised by malvidin-based anthocyanins, driven primarily by increases in the acylated derivatives. Under shade alone and under high temperature extremes in sunlit and shaded fruit, acylated anthocyanins represented a larger proportion of TSA than did nonacylated anthocyanins. At berry temperatures equivalent to those of shaded fruit, exposure to solar radiation decreased the proportion of TSA comprised by acylated forms of the five base anthocyanins and increased the proportion of TSA comprised by dihydroxylated anthocyanins. Results indicate a complex combined effect of solar radiation and berry temperature on anthocyanin composition, synergistic at moderate berry temperatures and potentially antagonistic at high temperature extremes.

Hhat Is a Palmitoylacyltransferase with Specificity for N-Palmitoylation of Sonic Hedgehog

Palmitoylation of Sonic Hedgehog (Shh) is critical for effective long- and short-range signaling. Genetic screens uncovered a potential palmitoylacyltransferase (PAT) for Shh, Hhat, but the molecular mechanism of Shh palmitoylation remains unclear. Here, we have developed and exploited an in vitro Shh palmitoylation assay to purify Hhat to homogeneity. We provide direct biochemical evidence that Hhat is a PAT with specificity for attaching palmitate via amide linkage to the N-terminal cysteine of Shh. Other palmitoylated proteins (e.g. PSD95 and Wnt) are not substrates for Hhat, and Porcupine, a putative Wnt PAT, does not palmitoylate Shh. Neither autocleavage nor cholesterol modification is required for Shh palmitoylation. Both the Shh precursor and mature protein are N-palmitoylated by Hhat, and the reaction occurs during passage through the secretory pathway. This study establishes Hhat as a bona fide Shh PAT and serves as a model for understanding how secreted morphogens are modified by distinct PATs.

Anthocyanins Present in Selected Tropical Fruits: Acerola, Jambolão, Jussara, and Guajiru

Many tropical fruits are rich in anthocyanins, though limited information is available about the characterization and quantification of these anthocyanins. The identification and quantification of anthocyanin pigments in four tropical fruits were determined by HPLC-MS/MS. Fruits studied included acerola (Malphigia emarginata), jussara (Euterpe edulis), jambolão (Syzygium cumini), and guajiru (Chrysobalanus icaco). All four fruits were found to contain anthocyanin pigments. Anthocyanidin backbones included cyanidin, delphinidin, peonidin, pelargonidin, petunidin, and malvidin. Guajiru contained several acylated forms, while acerola, jussara, and jambolão contained only nonacylated glycosides. These results demonstrate that these tropical fruits are rich in anthocyanins and that the anthocyanins are widely ranging in anthocyanidin backbone, glycosylation, and acylation.

Flavonoids in Horse Chestnut (Aesculus hippocastanum) Seeds and Powdered Waste Water Byproducts

Horse chestnut extracts are widely used in pharmacy and cosmetic industries. The main active constituents are saponins of oleane type, but seeds of horse chestnut also contain flavonoids, being glycosides of quercetin and kaempferol. Their contribution to the overall activity of the extracts was not clear. In the present work, the main flavonoids from horse chestnut seeds were isolated and their structures established with spectral methods. Seven glycosides were isolated, out of which six ( 2, 3, 4, 7, 11, 13) were previously reported and one ( 9) was identified as a new tamarixetin 3- O- [beta- d-glucopyranosyl(1-->3)]- O-beta- d-xylopyranosyl-(1-->2)- O-beta- d-glucopyranoside. The structures of three additional compounds 1, 10, and 12, not previously reported, were deduced on the basis of their LC-ESI/MS/MS fragmentation characteristics. A new ultraperformance liquid chromatographic (UPLC) method has been developed for profiling and quantitation of horse chestnut flavonoids. The method allowed good separation over 4.5 min. Thirteen compounds could be identified in the profile, out of which di- and triglycoisdes of quercetin and kaempferol were the dominant forms and their acylated forms occurred in just trace amounts. The total concentration of flavonoids in the powdered horse chestnut seed was 0.88% of dry matter. The alcohol extract contained 3.46%, and after purification on C18 solid phase, this concentration increased to 9.40% of dry matter. The flavonoid profile and their content were also measured in the horse chestnut wastewater obtained as byproduct in industrial processing of horse chestnut seeds. The total flavonoid concentration in the powder obtained after evaporation of water was 2.58%, while after purification on solid phase, this increased to 11.23% dry matter. It was concluded that flavonoids are present in a horse chestnut extract in a relatively high amount and have the potential to contribute to the overall activity of these extracts. Industrial horse chestnut wastewater can be used to obtain quercetine and kaempferol glycosides for cosmetic, nutraceutical, and food supplement industries.

Anthocyanins from red cabbage – stability to simulated gastrointestinal digestion

Anthocyanins were the main polyphenol components in extracts of fresh and pickled red cabbage. The composition of anthocyanins in red cabbage was studied using liquid chromatography mass–spectrometry. Eleven major peaks absorbing at 520 nm were discerned, which represented 18 different anthocyanin structures. Another five minor anthocyanin components could be identified by searching at their respective m/ z values but only in anthocyanin-enriched concentrates produced by sorption to solid phase extraction matrices. The predominant anthocyanins were constructed of cyanidin-3-diglucoside-5-glucoside “cores” which were non-acylated, mono-acylated or di-acylated with p-coumaric, caffeic, ferulic and sinapic acids. Pelargonidin-3-glucoside and novel forms of cyanidin-3- O-triglucoside-5- O-glucoside di-acylated with hydroxycinnamic acids were also detected in extracts of raw red cabbage, commercially pickled red cabbage and anthocyanin-enriched concentrates. The stability of the anthocyanins to simulated gastrointestinal digestion was assessed. The anthocyanins were effectively stable in the acidic gastric digestion conditions but the total recovery after simulated pancreatic digestion was around 25% compared to around 100% recovery of phenol content. As anthocyanins make up the majority of red cabbage polyphenols, this suggested that anthocyanins broke down to form new phenolic components. The recovery of the individual anthocyanins was monitored by LC–MS n . All of the anthocyanins were reduced in content after pancreatic digestion but acylated forms were notably more stable than non-acylated forms. There was also a relationship between the type of acylated hydroxycinnamic acid and stability to pancreatic digestion.

Gastrointestinal Uptake of Nasunin, Acylated Anthocyanin in Eggplant

We previously showed that nasunin, acylated anthocyanins in eggplant peel, comprises two isomers, cis-nasunin and trans-nasunin. In this study, gastrointestinal absorption of cis- and trans-nasunins was studied in rats. Orally administered nasunins were quickly absorbed in their original acylated forms and maximally appeared in blood plasma after 15 min. When the maximum plasma concentration and area under the plasma concentration curve were normalized by orally administered dose (micromoles per kilogram), there was no significant difference in the uptake efficiency between two isomers and both exhibited a plasma level almost identical to that of delphinidin 3-O-beta-D-glucopyranoside. However, metabolites such as 4'-O-methyl analogues and extended glucuronides which were observed for delphinidin 3-O-beta-D-glucopyranoside and cyanidin 3-O-beta-D-glucopyranoside metabolisms were not detected in urine or blood plasma. Moreover, deacylated and glycolytic products of nasunins such as delphinidin 3-O-beta-D-glucopyranoside or delphinidin (aglycone) were also not detected in blood plasma even after oral administration for 8 h. These results indicated that nasunins were absorbed in their original acylated forms and exhibit a bioavailability almost identical to that of nonacylated anthocyanins.

Non‐targeted profiling of lipids during kainate‐induced neuronal injury

Kainate is a glutamate analog that has been widely used in pharmacological studies of neuronal injury related to ischemic conditions and epilepsy. While altered lipid metabolism has been implicated in kainate action, no study has yet investigated the associated changes in lipid metabolites on a systems scale. Here we describe a mass spectrometry-based approach for profiling of lipid mixtures in a nontargeted fashion. Combined with tandem mass spectrometry, this method aims to identify lipids that are altered between two conditions, the kainate-treated and the control hippocampal tissues. In addition to reductions in major phospholipids with mainly polyunsaturated fatty acyl chains, we find elevated levels of ions that correspond to acylated forms of phosphatidylethanolamines and ceramides. Acylated phosphatidylethanolamines are neuroprotective lipids and precursors for anandamide, which signals via cannabinoid receptors. Quantitative analysis of ceramides shows that many molecular species with different acyl compositions are increased during kainate treatment. This increase is mainly restricted to neurons rather than other brain cells in the hippocampus as revealed by immunohistochemistry of brain slices.

Identification of immunoreactive plasma and stomach ghrelin, and expression of stomach ghrelin mRNA in the bullfrog, Rana catesbeiana

In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13–28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83–135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca 2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog.

Phenolic profiles of Andean purple corn ( Zea mays L.)

Qualitative and quantitative high performance liquid chromatography with diode array detection (HPLC-DAD) was performed to characterize the presence of phenolic compounds in Andean purple corn. Phenolic compounds were analyzed by separating them in two main fractions: a water fraction (WF) and an ethyl acetate fraction (EAF). The WF rich in anthocyanins revealed the presence of cyanidin-3-glucoside, pelargonidin-3-glucoside and peonidin-3-glucoside. The respective acylated anthocyanin glucoside forms of these compounds were also detected following alkaline hydrolysis. The EAF was composed of phenolic acids such as p-coumaric, vanillic acid, protocatechuic acid, flavonoids such quercetin derivatives and a hesperitin derivative. Alkaline and acid hydrolysis of the EAF revealed the presence of p-coumaric and ferulic acid as main components in four bound hydroxycinnamic acid forms present in the ethyl acetate fraction.

Preparation of isotopically labeled spinach acyl–acyl carrier protein for NMR structural studies

Acyl carrier proteins (ACPs) are important protein cofactors in fatty acid biosynthesis, but their acylated forms have not been well-studied. To permit detailed nuclear magnetic resonance studies of acylated spinach ACP isoform I, we have developed a new expression plasmid for recombinant production of the apo-protein and modified protocols for purifying the protein product and acylating it to form acyl-ACP. To solve plasmid stability problems associated with growth in minimal media, the ampicillin resistance gene from pSACP-2a was replaced with the tetA( C) gene from pBR322. The resulting plasmid, pSACP-2t, supported overexpression of apo-ACP in Escherichia coli BL21(DE3) cells in M9 medium containing 15NH 4Cl as the sole nitrogen source. Apo-ACP was purified to homogeneity by means of polyethylene glycol precipitation and anion exchange. Two in vitro synthetic routes were used to produce acyl–ACPs. In one route, apo-ACP was converted to the holo form and the acyl form by a published protocol that employs a discrete enzymatic reaction for each step. As an alternative route to produce decanoyl-ACP, apo-ACP was directly converted to the acyl form by using holo-ACP synthase along with the non-natural substrate decanoyl-CoA. Two-dimensional 1H– 15N NMR spectroscopy of decanoyl-ACP and stearoyl-ACP revealed that changes in the length of the covalently attached fatty acid do not affect the secondary structure of the protein but do influence the local conformation and dynamics.