Kinetic Inhibitors Research Articles

Experimental study of hydrogen sulfide hydrate formation: Induction time in the presence and absence of kinetic inhibitor

In this paper, the effect of adding different concentrations of kinetic inhibitors on the induction time of hydrogen sulfide hydrate formation in a reactor equipped with automatic adjustable temperature controller is studied. A novel method namely “sudden cooling” is used for performing the relevant measurements, in which the induction time of H2S hydrate in the presence/absence of PVP and L-tyrosine with different concentrations (100, 500, and 1000 ppm) is determined. As a result, PVP with the concentration of 1000 ppm in aqueous solution is detected as a more suitable material for increasing the induction time of H2S hydrate formation among the investigated kinetic hydrate inhibitors.

Study of Effects of Cigarette Smoke Condensates on Acetylcholinesterase Activity in Human Lung Epithelial Cells by Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Mass Spectrometry

Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Mass Spectrometry (MALDI-FTMS) is an emerging method for kinetic measurements and inhibitor screening of acetylcholinesterase (AChE). This method is accurate, reproducible, and was successfully applied to the analysis of various biosamples. Recent studies showed the important biological functions of acetylcholinesterase in many kinds of tumors caused by smoking; however, the effect of cigarette smoke condensates (CSCs) on AChE was unclear. Herein, the effects of CSCs on AChE activity in human lung epithelial cells were studied by MALDI-FTMS. The results showed that AChE activity in human lung epithelial cells could be inhibited by CSCs, and inhibition degree on AChE activity depended on the amount of CSCs. Owing to the close relationship of AChE to lung cancer caused by smoking, such findings provide information regarding the relation between the aggressiveness of lung tumor and cigarette smoking.

Bacterial Inhibition of Methane Clathrate Hydrates Formed in a Stirred Autoclave

The potential for hydrate inhibition in a stirred autoclave at a subcooling of 2.3 K has been tested on two bacterial isolates. Chryseobacterium sp. C14 survives multiple freeze–thaw events and inhibits ice recrystallization, and Escherichia coli has neither of these properties but is a biofilm producer. Both strains showed methane hydrate inhibition, with a significant reduction in total hydrate formed. Chryseobacterium delayed hydrate nucleating time, to a similar level found for the commercial kinetic inhibitor, polyvinylpyrrolidone (PVP; 0.2 wt %). However, in the presence of E. coli, in comparison to PVP, the time to nucleation was almost tripled and the hydrate growth rate was reduced by half. Because of the variation inherent with the microbial samples, likely a result of the complexities associated with varying cell numbers and metabolic activity, numerous experiments were required. Analysis of the distinct hydrate growth profiles shown by the two bacterial strains indicated that different molecul...

Kinetic Studies on Methane Hydrate Formation in the Presence of Kinetic Inhibitor via in Situ Raman Spectroscopy

To fully understand the influence of kinetic inhibitor, poly-N-vinylcaprolactam (PVCap), on methane hydrate formation, gas uptake measurement (macroscopic point of view) and in situ Raman spectroscopic analysis (microscopic point of view) were carried out simultaneously in a semibatch stirred tank reactor at constant temperature (T) and pressure (P). The capturing the behavior of guest molecules (large to small cavity ratio or cage variation) is one of the most important properties of gas hydrate studies. However the real-time properties of the cage variation under constant T and P conditions in an agitation system have not been reported previously. In this study, we measured this property (i.e., the large to small cavity ratio) from the in situ Raman spectra during hydrate formation in an agitation system instead of a static system, which provided valuable information on the time-dependent hydrate kinetic behavior. The study reveals that the presence of PVCap prevents the rate of large cavity encapsulati...

Assessing the performance of commercial and biological gas hydrate inhibitors using nuclear magnetic resonance microscopy and a stirred autoclave

The formation kinetics of methane/ethane/propane hydrate in the presence of kinetic inhibitors was investigated using 1H nuclear magnetic resonance imaging (MRI) as well as a more-traditional method using a stirred autoclave. These studies were facilitated by fabricating a multi-drop insert for 1H NMR micro-imaging, which allowed the comparison of the performance of microliter quantities of several inhibitors simultaneously and under the same conditions. Both methods showed that hydrate nucleation and growth were delayed significantly in the presence of inhibitors, which included two biological inhibitors (antifreeze proteins) and a commercial inhibitor. The results demonstrate that MRI is a useful tool for the visualization and evaluation of the performance of kinetic inhibitors on mixed gas hydrate formation. The MRI technique should prove especially valuable in the case of analysis of potential inhibitors, pre-commercialization, which are available in only limited quantities, such as biological inhibitors. This technique may also find utility in the exploration of differences in inhibitor performance, which may suggest distinct mechanisms of inhibitor action.

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via Förster Resonance Energy Transfer (FRET). The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

Involvement of the Penta-EF-Hand Protein Pef1p in the Ca2+-Dependent Regulation of COPII Subunit Assembly in Saccharomyces cerevisiae

Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca2+-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca2+-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca2+ dependence for Sec13/31p association; only the Ca2+-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca2+-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.

Allergen‐derived proteinases: Isolation, characterization and signaling via proteinase‐activated receptors (PARs)

ObjectiveTo identify proteolytic enzymes in insect and mould asthma allergens and assess their ability to signal via the PARs.MethodsAllergen‐derived trypsin‐like enzymes known to contribute to lung inflammation were visualized (western blot) with serine proteinase activity‐based probes (ABP). Enzymes were purified using ion‐exchange chromatography and analyzed for their ABP reactivity & substrate‐inhibitor kinetic profiles. Each enzyme was tested for its activation of PAR–dependent calcium signaling in PAR1 & PAR2‐expressing KNRK cells. ABP‐labeled enzymes were isolated by avidin affinity chromatography and their peptide sequences determined (mass spectrometry).ResultsBoth allergens contained ABP‐labeled proteinases with MWs of 20–26 kDa. Each enzyme had distinct kinetic substrate (Km) and inhibitor (Ki) profiles. The enzymes all caused PAR2 calcium signaling, and differentially activated PAR1. Sequencing revealed that the allergen proteinases were homologous with previously described trypsin‐like enzymes.ConclusionsDiverse allergens contain multiple biochemically distinct trypsin‐like proteinases that are able to target PARs 1 and 2. Thus, PARs and their activating allergen proteinases may be attractive therapeutic targets for the treatment of allergen‐induced asthma.Support: Canadian Institutes of Health Research, NIH USA and Lung Association of Alberta & NWT

Synthesis of Effective Kinetic Inhibitors for Natural Gas Hydrates

Six novel polymer-based kinetic hydrate inhibitors (KHIs) were synthesized and characterized. Their performance in inhibiting both tetrahydrofuran (THF) hydrate and the synthetic gas hydrate formation was examined using two different instruments: a ball-stop apparatus and a high-pressure automatic lag time apparatus (HP-ALTA). Performance was benchmarked against two commercially available KHIs, Gaffix VC-713 and Luvicap EG, under the same working conditions. The test results from the ball-stop rig demonstrated that the new KHIs were as effective as Gaffix VC-713 and Luvicap EG in preventing the formation of THF hydrates in 3.5 wt % NaCl solutions. For the synthetic gases, most new polymers outperformed the reference KHIs at a concentration of 0.05 wt %. Polymers containing a pendant THF functional group in the side chains showed a substantial 12–16 °C decrease in the hydrate formation temperature of the gas water mixtures relative to those containing the same amount of Gaffix VC-713 or Luvicap EG. The trend of the inhibition performance of the polymers was different in THF from that measured for gas mixtures. Small amounts of ethanol added to the hydrate formation mixtures were also shown to have an effect. Investigation of the inhibition mechanism associated with these new polymers is under way.

A polyether glycol derived from cashew nutshell as a kinetic inhibitor for methane hydrate formation

The polyether glycol derived from cashew nutshell liquid (CNSL) inhibited the formation of methane hydrate. The polymer proved to be more efficient than the polyvinyl pyrrolidone-poly(N-vinyl) caprolactam (PVP-PVCap) co-polymer under tested conditions (CH4, 1470 psi and 4 ºC), being the latter one of the best commercially available hydrate inhibitors.

Experimental Investigation of Natural Gas Components During Gas Hydrate Formation in Presence or Absence of the L-Tyrosine as a Kinetic Inhibitor in a Flow Mini-loop Apparatus

Hydrates are crystalline compounds similar to ice, with guest molecules like methane and ethane trapped inside cavities or cages formed by the hydrogen bounded framework of water molecules. These solid compounds give rise to problems in the natural gas oil industry because they can plug pipelines and process equipments. Low dosage hydrate inhibitors are a recently developed hydrate control technology, which can be more cost-effective than traditional practices such as methanol and glycols. The main objective of the present work is to experimentally investigate simple gas hydrate formation with or without the presence of kinetic inhibitors in a flow mini-loop apparatus. For this purpose, a laboratory flow mini-loop apparatus was set up to measure the induction time and gas consumption rate during gas hydrate formation when a hydrate forming substance such as methane, ethane, propane, carbon dioxide and iso- butane is contacted with water in the absence or presence of dissolved inhibitor at various concentration under suitable temperature and pressure conditions. In each experiment, a water blend saturated with pure gas is circulated up to a required pressure. Pressure is maintained at a constant value during experimental runs by means of the required gas make-up. The effect of pressure on gas consumption during hydrate formation is investigated with or without the presence of PVP (polyvinylpyrrolidone) and L-tyrosine as kinetic inhibitors at various concentrations. The experimental results show that increasing the pressure of the system, causes to increase the experimental gas consumption and decrease the induction time. Also, the extent of gas hydrate formation at a given time is clearly less in the presence of the inhibitors. Moreover, when comparing the gas consumption during the hydrate formation for simple gas hydrate formation in presence of PVP and L-tyrosine inhibitors, it is seen that the gas consumption in presence of L-tyrosine is lower than that of PVP for all experiments.

Natural Gas Hydrate Formation and Decomposition in the Presence of Kinetic Inhibitors. 3. Structural and Compositional Changes

A synthetic natural gas mixture composed of methane, ethane, and propane in a batch reactor was used to form gas hydrates in the presence of two commercial, chemical kinetic inhibitors, polyvinylpyrrolidone (PVP) and H1W85281, and one biological inhibitor, antifreeze protein type III (AFP-III). Powder X-ray diffraction and nuclear magnetic resonance spectroscopy showed that structure II hydrates dominated, as expected, but in the presence of the chemical inhibitors, structure I was also present. Raman spectroscopy confirmed the complexity and the heterogeneity of the guest composition within these hydrates, which was also consistent with the gas analysis obtained using gas chromatography. However, in the presence of AFP-III, hydrates appeared to be relatively homogeneous structure II hydrates, with weaker evidence of structure I. When individual gas cage occupancies were calculated, both classes of inhibitors reduced large cage methane occupancy by ∼25%. With the chemical inhibitors, these large cage meth...

Natural Gas Hydrate Formation and Decomposition in the Presence of Kinetic Inhibitors. 2. Stirred Reactor Experiments

A newly fabricated, stirred reactor was used to investigate hydrate inhibition and decomposition in the presence of two commercial, chemical kinetic inhibitors, polyvinylpyrrolidone (PVP) and H1W85281, as well as two antifreeze proteins (AFPs), type I and type III. The longest induction times and the slowest growth rates were observed with HIW8581, with the fastest growth recorded for PVP. Type I AFP (AFP-I) was a more effective inhibitor, with respect to induction time and growth, than either PVP or type III AFP (AFP-III). Complete hydrate decomposition occurred earlier in the presence of any of the inhibitors compared to water controls. However, depending on the type of inhibitor present during crystallization, hydrate decomposition profiles were distinct, with a longer, two-stage decomposition profile in the presence of the chemical kinetic inhibitors (PVP and H1W85281). The fastest, single-stage decompositions were characteristic of hydrates in experiments with either of the AFPs. These results argue ...

Natural Gas Hydrate Formation and Decomposition in the Presence of Kinetic Inhibitors. 1. High Pressure Calorimetry

The effect of kinetic inhibitors, both chemical (PVP and H1W85281) and biological (type III antifreeze protein), on natural gas hydrate formation was investigated using high pressure differential scanning calorimetry (HP-DSC). The presence of inhibitors decreased the overall formation of methane/ethane/propane hydrate compared to systems without added inhibitors. As well, all of the inhibitors significantly delayed hydrate nucleation as compared to water controls. However, the two classes of inhibitors were distinguished by the formation of hydrates with different stabilities. A single hydrate melting peak was seen with the antifreeze protein (AFP), and this was consistent after recrystallization. In contrast, multiple hydrate melting events, some indicating the formation of hydrate structures with high stability, were observed in the presence of the chemical inhibitors, and these varied depending on the crystallization cycle. This heterogeneity suggests that the use of these chemical inhibitors (PVP and ...

Experimental investigation of double gas hydrate formation in the presence of modified starch as a kinetic inhibitor in a flow mini‐loop apparatus

AbstractThe main objective of the present work is experimental investigation of double gas hydrate formation with or without presence of modified starch as kinetic inhibitors in a flow mini‐loop apparatus. To this object, a laboratory flow mini‐loop apparatus was set up to measure the induction time for hydrate formation and the rate uptake when a gaseous mixture (such as 75% C1–25% C3, 25% C1–75% C3, 75% C1–25% i‐C4 and 25% C1–75% i‐C4) is contacted with water containing or not containing dissolved inhibitor under suitable temperature and pressure conditions. In each experiment, water blend saturated with gas mixture is circulated up to a required pressure. Pressure is maintained at a constant value during experimental runs by means of required gas mixture make‐up. The effect of pressure on gas consumption during hydrate formation is investigated with or without presence of polyvinylpyrrolidone and modified starch as kinetic inhibitors at various concentrations. Our results were shown that the modified starch can be applied as inhibitors in prevention of double gas hydrate formation in mini‐loop apparatus. © 2011 Canadian Society for Chemical Engineering

Enhancement of the performance of kinetic inhibitors in the presence of polyethylene oxide and polypropylene oxide for binary mixtures during gas hydrate formation in a flow mini‐loop apparatus

AbstractThe objective of this work is to demonstrate the impact of the polyethylene oxide (PEO) and polypropylene oxide (PPO) on the performance of gas hydrate kinetic inhibitors for binary mixtures during gas hydrate formation in a flow mini‐loop apparatus. PEO and PPO are commercially available polymers that they have been considered to be unable to exhibit kinetic hydrate inhibition (KHI) by their self. Prevention of gas hydrate formation experiments in the presence of the KHIs solutions were conducted in a flow mini‐loop apparatus manner under suitable pressures and temperature conditions for binary gaseous mixtures including 70% CH4/30% C3H8, 30% CH4/70% C3H8, 70% CH4/30% i‐C4H10, and 30% CH4/70% i‐C4H10. In the experiments, induction time for crystallisation of gas hydrate formation and gas consumption rate are investigated in systems without KHI, containing KHI only (such as polyvinylpyrrolidone (PVP) and L‐tyrosine) and PEO or PPO together with KHI. Pressure is maintained at a constant value during experimental runs by means of required gas make‐up. The addition of a KHI into system delayed the onset of hydrate crystal nucleation. Furthermore, addition of the PEO or PPO to a KHI solution was found to enhance the performance of KHI. In addition, under the same pressure temperature hydrate formation conditions the induction time is longer when the PPO is present. Thus, inclusion of PPO into a KHI solution shows a higher enhancement in its inhibiting performance compare to PEO. © 2011 Canadian Society for Chemical Engineering

Homo- and Copolymers of N-Acryloylpyrrolidine and N-Vinylpyrrolidone as Kinetic Inhibitors of Hydrate Formation

Two copolymers of N-acryloylpyrrolidine and N-vinylpyrrolidone with a different ratio of monomeric units and two homopolymers; poly-N-acryloylpyrrolidine and poly-N-vinylpyrrolidone, were synthesized to study the effect of the composition of macromolecular compounds (MMC) on their inhibiting power during formation of gas hydrates. The composition of the MMC was confirmed by 1H and 13C NMR spectroscopy and the ratio of monomeric units in the copolymers was determined with the 1H NMR spectra. The inhibiting power of the synthesized MMC was determined during hydrate formation.

Inhibition of Structure I and II Gas Hydrates using Synthetic and Biological Kinetic Inhibitors

Use of low-dosage hydrate inhibitors in oil and gas field applications has been limited mainly because there are uncertainties related to their efficiency in preventing hydrate formation or because of environmental restrictions. Another question that normally arises in connection with use of low-dosage hydrate inhibitors is how they interact with other (than water) compounds normally found in oil and gas production pipelines. In this work three different experimental approaches to obtain hydrate induction times have been tested for structure I and II hydrate. All experiments were conducted in a high-pressure stirred cell. The results suggest that by adding small amounts of impurities to the hydrate-forming system a significant improvement in the reproducibility of induction times can be obtained. Adding salt and heptane to the system (as a simple model for seawater and a light crude) was found to increase the hydrate formation rate. Ice-structuring protein type III identified in the ocean pout, a biologic...

Characterization of inhibition mechanisms of kinetic hydrate inhibitors using ultrasonic test technique

The inhibition characteristics of kinetic hydrate inhibitors were experimentally investigated by utilization of the ultrasonic testing technique. PVCap (poly(vinylcaprolactam)) and VP/BA (vinyl pyrrolidone/butyl acrylate) were chosen as two typical kinetic inhibitors. Results of the tests demonstrate two kinds of inhibition mechanisms of kinetic hydrate inhibitors, i.e., nucleation inhibition and growth inhibition. PVCap polymer is strong to retard catastrophic growth of hydrate formation, while VP/BA dominantly prevents hydrate nucleation. The synergism of glycol ether compounds on the performance of PVCap was also tested. It was found that glycol ether compounds noticeably prolong the nucleation time, and extend the delay of catastrophic growth significantly.