Kinetic Inhibitors Research Articles

Microbial dolomite precipitation using sulfate reducing and halophilic bacteria: Results from Qinghai Lake, Tibetan Plateau, NW China

Past studies have revealed the important roles of certain types of microbes in precipitating dolomite in coastal hypersaline lakes and lagoons, and marine sediments, however, it is still poorly known if other types of microbes can precipitate dolomite in low-salinity lakes without any marine influence. The objective of this study was therefore to expand the microbial dolomite precipitation model by investigating the origin of dolomite in the sediments of Qinghai Lake, an inland, slightly saline lake (salinity 12.5 g/L) on the Tibetan Plateau, NW China. This objective was accomplished via an integrated approach combining geochemical investigations of natural dolomite present in the lake sediments with laboratory precipitation experiments. A 5-m sediment core was collected and pore water chemistry was analyzed. Calculation of saturation index indicated that dolomite was oversaturated in sediment pore water. Sub-spherical grains of dolomite with abundant pores and microstructures, similar to microbially precipitated dolomite, were found in the sediments, suggesting a biogenic origin. Dolomite precipitation experiments were set up in the laboratory to simulate the natural conditions of the Qinghai Lake. Dolomite was precipitated using sulfate reducing bacterium (SRB) Desulfotomaculum ruminis and aerobic halophilic bacterium Halomonas marina present in the Qinghai Lake sediments. Aqueous geochemistry and mineral characterization data collectively suggest that dolomite precipitation was accomplished via increase of pH and alkalinity to overcome the kinetic inhibitors and exopolymeric substances (EPS) and cell walls as possible nucleation sites. These results expand the current microbial dolomite precipitation model and have implications for dolomite formation in slightly saline, inland lake environments.

Anti-agglomeration in Cyclopentane Hydrates from Bio- and Co-surfactants

Hydrate formation in subsea pipelines is a serious problem in gas and oil production for offshore fields. Current methods are mainly based on thermodynamic inhibitors to change bulk phase properties. Thermodynamic inhibitors, such as methanol, are very effective, but large quantities, sometimes as high as a 1:1 volume of alcohol/water, are required. Kinetic inhibitors generally in a 0.005−0.02 volume ratio of surfactant/water can either inhibit hydrate formation or reduce the rate of growth. In the sea bed, the subcooling for hydrates is around 20−25 °C because of the sea bed temperature of about 4 °C. The kinetic inhibitors are not effective at such a high subcooling. An effective method is the use of anti-agglomerants, which allow for hydrate formation in the form of small particles and prevent agglomeration of such particles. Rhamnolipid biosurfactant and methanol are used recently to demonstrate anti-agglomeration in tetrahydrofuran (THF) hydrates. In this work, we present data for cyclopentane hydrat...

Assessment of hydrate kinetic inhibitors with visual observations

The hydrate inhibition effect of three kinetic inhibitors (inhibex 301, 501, and 713) was assessed from (CH 4 + C 2H 6 + C 3H 8) gas mixture + brine systems using a high pressure sapphire cell. The onset time of hydrate formation was determined by visual observation method and pressure drop profile method, respectively. The experimental results demonstrated that the onset time was able to be determined by the visual observation method all the time while the pressure drop profile method failed to detect the onset time clearly and correctly at lower temperatures. In some cases, the initial appearance of hydrate crystals cannot induce a clear break in the pressure–time relationship curve. The onset time measured by the visual observation method is usually shorter than or at least the same as that determined by the pressure drop profile method. The inhibiting effect on the growth of hydrate crystals can be shown by the difference of the onset time obtained by the two methods. The maximum tolerated subcooling of each inhibitor was also investigated based on the onset time. It was found that inhibex 301 behaves as the best inhibitor that can tolerate the maximum subcooling of 8.3 K at 0.5 wt% and 10.6 K at 1.0 wt%, respectively. The maximum subcooling for inhibex 501 is 6.8 K at 0.5 wt% and 6.6 K at 1.0 wt%, respectively. Inhibex 713 has relatively poor inhibiting effect among the three inhibitors with the maximum subcooling of less than 3.5 K at 0.5 wt% and 5.1 K at 1.0 wt%, respectively.

Chitosan as green kinetic inhibitors for gas hydrate formation

The kinetic inhibiting effect of a number of chitosans on hydrate formation was investigated using methane and methane/ethane gas mixtures. The results indicated that chitosan was a good kinetic inhibitor. The induction time of gas hydrate formation evidently increased with the degree of deacetylation (DD), however, when DD was higher than 80%, the effect of DD on the induction time was negligible. Moreover, it was found that the molecular weight (MW) of chitosan and the addition of polyethylene oxide (PEO) had little effect on the induction time. The optimal concentration of chitosan was found to be 0.6 wt%. Finally, the mechanisms of the kinetic inhibitor on the hydrate formation were discussed.

Bile Acids Stimulate ATP Hydrolysis in the Purified Cholesterol Transporter ABCG5/G8

ABCG5 and ABCG8 are half-size ABC transporters that function as heterodimers (ABCG5/G8) to reduce sterol absorption in the intestines and increase sterol excretion from the liver. Previous studies demonstrated that bile acids increased ABCG5/G8 specific cholesterol efflux in cell models. In this study we tested the effects of bile acids on ATP hydrolysis in Pichia pastoris purified ABCG5/G8 and found that they stimulated hydrolysis approximately 20-fold in wild-type ABCG5/G8 but not in a hydrolysis-deficient mutant. Nonconjugated cholate supported the highest ATPase activity in ABCG5/G8 (256 +/- 9 nmol min(-1) mg(-1)). ATP hydrolysis was also stimulated by other conjugated bile acids and a mixture of bile acids resembling human bile with activities ranging from 129 +/- 4 to 147 +/- 14 nmol min(-1) mg(-1). The kinetic parameters, inhibitor profiles, and lipid requirements of bile acid stimulated ATP hydrolysis were characterized. Cholate-stimulated ATP hydrolysis was maximal at concentrations of >or=10 mM MgATP and had a relatively high K(M) (MgATP) of approximately 1 mM. Orthovanadate, BeFx, and AlFx effectively inhibited ABCG5/G8 at concentrations of 1 mM. Various lipid mixtures supported bile acid-stimulated ATP hydrolysis, which increased when cholesterol was present. The data demonstrate that bile acids together with lipids and cholesterol increase ATP hydrolysis in purified ABCG5/G8. Bile acids may promote an active conformation of purified ABCG5/G8 either by global stabilization of the transporter or by binding to a specific site on ABCG5/G8.

Nucleotide Selectivity of Antibiotic Kinases

Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3')-IIIa, APH(2'')-Ib, and MPH(2')-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3')-IIIa exclusively uses ATP, MPH(2')-I exclusively uses GTP, and APH(2'')-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.

Towards a Green Hydrate Inhibitor: Imaging Antifreeze Proteins on Clathrates

The formation of hydrate plugs in oil and gas pipelines is a serious industrial problem and recently there has been an increased interest in the use of alternative hydrate inhibitors as substitutes for thermodynamic inhibitors like methanol. We show here that antifreeze proteins (AFPs) possess the ability to modify structure II (sII) tetrahydrofuran (THF) hydrate crystal morphologies by adhering to the hydrate surface and inhibiting growth in a similar fashion to the kinetic inhibitor poly-N-vinylpyrrolidone (PVP). The effects of AFPs on the formation and growth rate of high-pressure sII gas mix hydrate demonstrated that AFPs are superior hydrate inhibitors compared to PVP. These results indicate that AFPs may be suitable for the study of new inhibitor systems and represent an important step towards the development of biologically-based hydrate inhibitors.

Inhibition of Methane Hydrate Formation by Ice-Structuring Proteins

In the oil and gas industry there is ample motivation for moving toward greener kinetic inhibitors of gas hydrates as many of those used today suffer from poor biodegradability. In this work, we have investigated experimentally whether ice-structuring proteins (ISPs) found in fish and insect, assumed biodegradable, are capable of inhibiting the growth of methane hydrate (a structure I hydrate). The ISPs investigated were type III HPLC12 (originally identified in ocean pout) and ISP type III found in meal worm (Tenebrio molitor). These were compared to polyvinylpyrrolidone (PVP) a well-known kinetic hydrate inhibitor. The results revealed that adding ISP in sufficient amounts caused the appearance of an initial nonlinear growth period. At a certain point during the growth process the growth pattern changed to linear which is identical to the growth observed for methane hydrate in the absence of inhibitors. The profile of the nonlinear growth was concentration-dependent but also dependent on the stirring rate. ISP type III HPLC12 decreased the growth rate of methane hydrate during the linear growth period by 17−75% at concentrations of 0.01−0.1 wt % (0.014−0.14 mM) while ISP from Tenebrio molitor and PVP decreased the growth rate by 30% and 39% at concentrations of 0.004 wt % (0.005 mM) and 0.1 wt % (0.1 mM), respectively. Considering the low concentration of Tenebrio molitor ISP used, these results indicate that ISP from Tenebrio molitor is the most effective hydrate inhibitor among those investigated. Thermal hysteresis ice formation experiments revealed that ISP from Tenebrio molitor causes higher thermal hysteresis for ice formation compared to type III ISP identified in ocean pout while PVP did not cause thermal hysteresis. This indicates that there might be a direct relationship between ISP performance for ice and hydrate inhibition, and that thermal hysteresis experiments can be used to screen ISPs as kinetic inhibitors.

THE EFFECT OF VARIOUS TYPES OF EQUATIONS OF STATE ON DRIVING FORCE CALCULATION AND GAS CONSUMPTION PREDICTION IN SIMPLE AND DOUBLE HYDRATE FORMATION WITH OR WITHOUT THE PRESENCE OF KINETIC INHIBITORS IN BATCH SYSTEMS

This article compares the effects of using various types of equations of state (Peng-Robinson, PR; Soave-Redlich-Kwong, SRK; Esmaeilzadeh-Roshanfekr, ER; Patel-Teja, PT; and Valderrama-Patel-Teja, VPT) on the calculated driving force and rate of gas consumption based on the Kashchiev model in simple and double-gas hydrate formation for methane, ethane, and their mixtures with 1130 experimental published data points with or without the presence of kinetic inhibitors at various pressures and temperatures. For the prediction of gas consumption rate in double-gas hydrate formation, the rate equation based on the Kashchiev model for simple gas hydrate formation was developed using the calculation of gas mole fraction in hydrate phase and then prediction of gas hydrate formation rate for each component in gaseous mixture. The total average absolute deviation was found to be 8.72%, 10.34%, 8.84%, 11.04%, and 14.16% for the PR, ER, SRK, VPT, and PT equations of state for calculating gas consumption in simple and double hydrate formation, respectively.

Dynamic surface tension studies on poly( N-vinylcaprolactam/ N-vinylpyrrolidone/ N, N-dimethylaminoethyl methacrylate) at the air–liquid interface

The adsorption of poly( N-vinylcaprolactam/ N-vinylpyrrolidone/ N, N-dimethylaminoethyl methacrylate), a commercial product with the trade name of Gaffix ® VC-713 and currently used as a kinetic inhibitor for natural gas hydrates, has been experimentally studied at the air–liquid interface using surface tension measurements. The influence of polymer concentration and temperature on the adsorption of the polymer was analysed. The equilibrium experimental data obtained at both 293 K and 278 K has been fitted using the analogous surface equation of state for the Langmuir isotherm. Dynamic adsorption has been modelled using both diffusion-controlled and asymptotic models. The values of the surface tension and the surface excess at saturation showed that the polymer was very active at the air–liquid interface. Equilibrium surface tension indicated that temperature has a weak influence on the number of moles of Gaffix ® VC-713 adsorbed on the interface. The dynamic adsorption was controlled by diffusion process initially, in which a rapid reduction in surface tension was observed. It was then followed by a slow polymer reorganization process. A strong dependence of the dynamic adsorption on both the temperature and the polymer concentrations was well demonstrated.

Intensification of the performance of kinetic inhibitors in the presence of polyethylene oxide and polypropylene oxide for simple gas hydrate formation in a flow mini-loop apparatus

The main objective of the present work is enhancement of the performance of gas hydrate kinetic inhibitors in the presence of polyethylene oxide (PEO) and polypropylene oxide (PPO) for simple gas hydrate formation in a flow mini-loop apparatus. PEO and PPO are high molecular weight polymers that are not kinetic inhibitors by their self. For this investigation, a laboratory flow mini-loop apparatus was set up to measure the induction time and rate of gas hydrate formation when a hydrate-forming substance (such as C1, C3, CO 2 and i-C4) is contacted with water containing dissolved inhibitor in presence or absence of PEO or PPO under suitable temperature and pressure conditions. In each experiment, water containing inhibitors 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 required gas make-up. The effect of PEO and PPO on induction time and gas consumption during hydrate formation is investigated in the presence or absence of PVP (polyvinylpyrrolidone) and l-tyrosine as kinetic inhibitors. Results were shown that the induction time is prolonged in the presence of PEO or PPO compared to the inhibitor only. Inclusion of PPO into a kinetic hydrate inhibitor solution shows a higher enhancement in its inhibiting performance compare to PEO. Thus, the induction time for simple gas hydrate formation in presence of kinetic hydrate inhibitor with PPO is higher, compare to kinetic hydrate inhibitor with PEO.

Kinetic Mechanism and Inhibitor Characterization of WNK1 Kinase

Pseudohypoaldosteronism type II (PHAII) is caused by the mutation of two members of the WNK (with-no-K[Lys] kinase) kinase family. We describe here the development of an in vitro WNK1 microfluidic mobility shift assay for kinetic mechanism studies. Assays using capillary electrophoresis on a microfluidic chip are suitable for both compound selection and mechanistic studies, because of the robustness of this method, as well as its high-throughput feature and insensitivity to the ATP concentration. Double-reciprocal plots of the initial rates versus the concentration of the substrate revealed that the random sequential activity of WNK catalyzed OXSR1 (oxidative stress response kinase-1) phosphorylation. WNK1 inhibitors were then found from among 86 kinases in a commercially available library. Interestingly, the Hck, Lck, and Src inhibitors, PP1 and PP2, exhibited positive inhibition against WNK1. The inhibition mode of PP1 was analyzed to be pure ATP competition with a K(i) value of 12.7 microM, showing noncompetitive inhibition against the OXSR1 peptide. From the structure-based comparison, we found that, since the WNK1 enzymes are categorized as STEs (homologues of yeast Sterile 7, Sterile 11, and Sterile 20 kinases) and Hck belongs to the TK (tyrosine kinase) family on the basis of the results of the Human Kinome Project, the residues at the catalytic site of the WNK1 that interact with PP1 were well-conserved in Hck. We concluded that the compound-based structural alignment enabled us to find interesting relationships among the kinases. This information helps us to screen specific WNK1 therapeutic reagents with no inhibition of the Src, Hck, and Lck kinases for the treatment of hypertension.

Adsorption of Kinetic Inhibitors on Clathrate Hydrates

Adsorption studies of kinetic inhibitors on clathrate hydrates are essential for understanding the inhibition mechanism at the hydrate−water interface. This work presents the adsorption behaviors of two kinetic inhibitors, polyvinyl pyrrolidone (PVP) and polyvinylcaprolactam (PVCap), on cyclopentane (CP) hydrates. The particle charge of CP hydrates is negative in the absence of any inhibitors, and it becomes neutral as the inhibitor concentration increases. The isotherm of PVP is well fitted to the Langmuir-type whereas the adsorption isotherm of PVCap is of the BET-type. Although PVCap monomers preferably adsorb on hydrates compared with PVP monomers, the isotherms of PVP and PVCap overlap with each other at concentrations up to 50 μM that corresponds to a water-based weight fraction of 0.6% for PVP or 0.5% for PVCap. Above this concentration, a multilayer adsorption occurs for PVCap. This multilayer adsorption gives rise to a thicker adsorption layer than that for the PVP case, which makes PVCap more effective in reducing the diffusion of hydrate formers from the bulk phase to the hydrate surface where the hydrate growth prefers to proceed. The determination of polymer inhibitor adsorption isotherms can provide the implication of adsorption behaviors and can be utilized to screen a new effective kinetic hydrate inhibitor.

Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme’s significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a K m of 46 μM and a k cat of 3.5 s −1 with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a K i of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.

Effect of various types of equations of state for prediction of simple gas hydrate formation with or without the presence of kinetic inhibitors in a flow mini-loop apparatus

This paper compares the effects of using various types of equations of state (PR, 1 1 Peng–Robinson. SRK, 2 2 Soave–Redlich–Kwong. ER, 3 3 Esmaeilzadeh–Roshanfekr. PT 4 4 Patel–Teja. and VPT 5 5 Valderrama–Patel–Teja. ) on the calculated driving force and rate of gas consumption based on the Kashchiev and Firoozabadi model for simple gas hydrate formation for methane, carbon dioxide, propane and iso-butane with experimental data points obtained in a flow mini-loop apparatus with or without the presence of kinetic inhibitors at various pressures and specified temperatures. For this purpose, a laboratory flow mini-loop apparatus was set up to measure gas consumption rate when a hydrate forming substance (such as C 1, C 3, CO 2 and i-C 4) is contacted with water in the presence or absence of dissolved inhibitor 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 total average absolute deviation was found to be 15.4%, 16.3%, 15.8%, 17.8% and 17.4% for the PR, ER, SRK, PT and VPT equations of state for calculating gas consumption in simple gas hydrate formation with or without the presence of kinetic hydrate inhibitors, respectively. Comparison results between the calculated and experimental data points of gas consumption were obtained in flow loop indicate that the PR and ER equations of state have lower errors than the SRK, VPT and PT equations of state for this model.

Fragment-based lead generation: identification of seed fragments by a highly efficient fragment screening technology

For the detection of the precise and unambiguous binding of fragments to a specific binding site on the target protein, we have developed a novel reporter displacement binding assay technology. The application of this technology for the fragment screening as well as the fragment evolution process with a specific modelling based design strategy is demonstrated for inhibitors of the protein kinase p38alpha. In a fragment screening approach seed fragments were identified which were then used to build compounds from the deep-pocket towards the hinge binding area of the protein kinase p38alpha based on a modelling approach. BIRB796 was used as a blueprint for the alignment of the fragments. The fragment evolution of these deep-pocket binding fragments towards the fully optimized inhibitor BIRB796 included the modulation of the residence time as well as the affinity. The goal of our study was to evaluate the robustness and efficiency of our novel fragment screening technology at high fragment concentrations, compare the screening data with biochemical activity data and to demonstrate the evolution of the hit fragments with fast kinetics, into slow kinetic inhibitors in an in silico approach.

Insulin is a kinetic but not a thermodynamic inhibitor of amylin aggregation

One of the most important pathological features of type 2 diabetes is the formation of islet amyloid, of which the major component is amylin peptide. However, the presence of a natural inhibitor such as insulin may keep amylin stable and physiologically functional in healthy individuals. Some previous studies demonstrated that insulin was a potent inhibitor of amylin fibril formation in vitro, but others obtained contradictory results. Hence, it is necessary to elucidate the effects of insulin on amylin aggregation. Here we report that insulin is a kinetic inhibitor of amylin aggregation, only keeping its inhibitory effect for a limited time period. Actually, insulin promotes amylin aggregation after long-term incubation. Furthermore, we found that this promotional effect could be attributed to the copolymerization of insulin and amylin. We also found that insulin copolymerized with amylin monomer or oligomer rather than preformed amylin fibrils. These results suggest that the interaction between insulin and amylin may contribute not only to the inhibition of amylin aggregation but also to the coaggregation of both peptides in type 2 diabetes.

Morphology Study of Structure I Methane Hydrate Formation and Decomposition of Water Droplets in the Presence of Biological and Polymeric Kinetic Inhibitors

The effect of kinetic inhibitors on the morphology of methane structure I hydrate was observed using a high pressure sapphire crystallizer. Two kinetic inhibitors were studied, poly(VP/VC), a lactam ring copolymer of polyvinylpyrrolidone (PVP) and polyvinylcaprolactam (PVCap), and type-I antifreeze protein (AFP). The experiments were performed at temperatures ranging from 274.2 to 275.2 K and pressures from 4200 to 7200 kPa. The experiments were conducted on three droplets simultaneously (a pure water droplet, a droplet containing 0.01 mol/m3 poly(VP/VC), and a droplet containing 0.01 mol/m3 AFP). The morphology and translucency were compared and found to vary significantly due to the presence of kinetic inhibitors. Hydrates formed under a higher driving force had dendrite formation on all but the AFP droplet. Low driving force experiments produced noticeably smoother surfaces on all droplets compared to high driving force experiments. Translucency also varied with AFP and poly(VP/VC) having the appearanc...

Evaluating the utility of the HTRF ® Transcreener ™ ADP assay technology: A comparison with the standard HTRF assay technology

The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay technology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detecting the formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we compare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as a case study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constants and inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic window and lower Z′ factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect both kinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenues for assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this can speed up the drug discovery process. However, caution must be exercised because any contaminating ATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP in the assay will also limit the application of this technology, especially in compound mechanistic studies such as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool to complement existing assay technologies for drug discovery.

Experimental and Theoretical Investigation of Double Gas Hydrate Formation in the Presence or Absence of Kinetic Inhibitors in a Flow Mini‐Loop Apparatus

AbstractDouble gas hydrate formation in the presence or absence of kinetic inhibitors in a flow mini‐loop apparatus was investigated. For the prediction of the gas consumption rate during hydrate formation in this system, the rate equation based on the Kashchiev and Firoozabadi model for simple gas hydrate formation in a batch system was developed for double gas hydrate formation in a flow mini‐loop apparatus. To complete the theoretical evaluation of gas hydrate formation through the mini‐loop apparatus in the presence or absence of kinetic hydrate inhibitors (KHI), a laboratory flow mini‐loop apparatus was set up to measure the induction time for hydrate formation and the uptake rate 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, a water blend saturated with gas mixture was circulated up to the required pressure. The pressure was maintained at a constant value during the experimental runs by means of a required gas mixture make‐up. The effect of pressure on gas consumption during hydrate formation was investigated in the presence or absence of polyvinylpyrrolidone (PVP) and L‐tyrosine as kinetic inhibitors at various concentrations. A good agreement was found between the predicted and experimental data in the presence or absence of KHI. The total average absolute deviation percents between the experimental and predicted values of gas consumption were found to be 16.4 and 17.5 % for the double gas hydrate formation in the presence or absence of the kinetic inhibitors, respectively.