The robustness of the method was analyzed by estimating the response obtained by deliberately changing the conditions. [Crossref]. 37% (w/w) HCl (PN: 131020) was purchased from Panreac (Barcelona, Spain). [Crossref], 4 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. In all cases, a sample blank was analyzed to demonstrate that there were no matrix interferences caused by the extract. The intensification of amyloglucosidase-based saccharification by ultrasound. Xiao Z., Storms R., Tsang A. Corrigendum to A quantitative starchiodine method for measuring alpha-amylase and glucoamylase activities [Anal. R. P.; Mol. Hi, Laneesha Moozhikkal This link can be helpful for the calculation of alpha amylase activity. http://www.microbiology.biology.upatras.gr/en/proto The vial was then placed for 10min into a heating block at 45C. Their age ranged between 25 65 years. This assay was also very precise, with RSD values below 3% in all cases. The analytical protocol can also be conducted in mild conditions (room temperature), bypassing the heating steps and the protocol complexity that is present in the DNS method, as well in other alternative methods for the same analytes [6]. 2017, Article ID 7319504. This corresponds to a total mass of 483.4g of glucose (mglucose) in the 3200L total volume. ASSESSMENT OF -AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD. [Crossref], 3 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. 1959, 31, 426. Enzymatic Method for Determining Amylase Activity (Amylase Activity Assay) This assay protocol is suitable for the colorimetric detection of Amylase activity in cell and tissue culture In our particular case [8], this study complemented our previous assessment of enzyme activity by the starch-iodine method. Standards for quantification of glucose yield. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National funds (FCT through grant no. An official website of the United States government. Get this protocol in PDF format. 1 ml of D.N.S. If necessary, the solution should be prepared fresh for daily use. [Crossref]. [Crossref], 11 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. One milligram of each extract was weighed and dissolved in 1 mL of phosphate buffer (100 mmol L-1, pH 6.9); some solutions were sonicated to achieve complete dissolution. This article will discuss the different applications of amylase. Because crude extract contains a variety of bioactive metabolites, it is necessary to characterize the bioactive compound showing inhibition activity by purifying it to determine its IC50 value. 1959, 31, 426. 17 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. [Crossref]) were used, and the experiment was conducted in triplicate. ( 2016 ). Disclaimer, National Library of Medicine The use of soluble starch also allowed the standardization of the working conditions, avoiding any influence of the solubility of the starch in the final result. Master degree dissertation. Plate uniformity assessment is mandatory for all assays. J. xZmoEna?T Therefore, the glucose yield corresponded to the ratio between mglucose (483.4g) and the total mass of starch mstarch (2400g), which resulted in a value of 20.14%. Amber glass vials (15mm45mm8mm, e.g. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980. Modern Agricultural Sciences and Technology. 5 ml of deionized water are added in each sample, followed by Here we introduced an improved method capable to measure the activity of alpha-amylase and amyloglucosidase from different sources based on a quantitative starch-iodine assay. PEst-C/EQB/LA0006/2011) and co-financed by FEDER (UID/QUI/50006/2013 NORTE-01-0145-FEDER-00011) under the Partnership Agreement PT2020. An official website of the United States government. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. Among solvents, ethanol (up to 25%) and DMSO (up to 0.5%) at lower concentrations did not have a significant out-turn, indicating that the method is robust for the abovementioned conditions. All experimental details are described in the section Amylolytic enzyme activity assay. Epub 2015 Feb 16. This study has explored methods that produce an efficient in vitro culture protocol for the conservation of Universidade Federal da Bahia. J. Mol. Regarding reagents, we describe in detail the preparation of the solutions of soluble starch and triiodide (from the KI / I2 solution), which are key elements for the starch-iodine method. Amylase is an enzyme responsible for breaking down carbohydrates, it is found in saliva and pancreatic juice, and it helps the body digest food. System Appl Microbiol 41:516527, Shaik M, Sankar GG, Iswarya M, Rajitha P (2017) Isolation and characterization of bioactive metabolites producing marine Streptomyces parvulus strain sankarensis-A10. The enzyme activity should be determined within one hour. These parameters should be adjusted according to the objectives of each individual study). The .gov means its official. 8600 Rockville Pike The new method facilitates rapid screening of a large number of samples, reduces labor, time and reagents and is also suitable for kinetic studies. The plant extract solutions were in the concentration range of 6 g mL-1 to 200 g mL-1. Soluble starch (PN: S9765), starch from corn (PN: S4126), starch from potato (PN: S4251), starch from rice (PN: S7260), starch from wheat (PN: S5127, unmodified), I2 (PN: 207772, 99.8%), 85% (w/w) o-phosphoric acid (PN: 79620), and acetic acid (PN: 695092, 99.7%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bookshelf Careers, REQUIMTE, LAQV, ICBAS, Instituto de Cincias Biomdicas de Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal. Food Sci. 2005 Mar;10(3):130-7. doi: 10.1016/j.tplants.2005.01.001. To achieve a good assay, there must be good agreement between the in vivo and in vitro results for the evaluated substance. In general, they have a poor design of experiments and an inadequate statistical significance.1111 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. where Xt is the added concentration of acarbose (the expected or true value of acarbose) and Xi is the experimentally determined concentration of acarbose. In each plate, the location of each solution was changed with respect to its column or row of wells. 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. The higher is the intensity of color, the higher is the concentration of reducing sugars in the solution. Thank you Marsha for your replay, actually I am trying to calculate IC50 or EC50 from my samples (plant extracts) to see if it can use as inhibitor [Crossref] This state of hyperglycemia can cause complications such as nephropathy, retinopathy, atherosclerosis, and memory impairment.22 de Sales, P. M.; de Souza, P. M.; Dartora, M.; Resck, I. S.; Simeoni, L. A.; Fonseca-Bazzo, Y. M.; de Oliveira-Magalhes P.; Silveira, D.; Food Chem. Toxicol. }$q6/vo~\eT9 Qu|Sn\tY?n}O65nnu:jqbWNVY6/`By[%"*"/^ry ~0*zp1= >Iu*#OR2ou8#jV|6i)UJ*G2ZE Wm^nVd~TaswO],+:h;, Screening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot Spring, International Journal of Science and Research (IJSR). Acarbose, an inhibitor of the amylase enzyme, was evaluated at six different concentration levels by regression analysis. We used a US bath to facilitate the complete solubilization of I2 and subsequent formation of triiodide (please note that the concentration of KI described in the original report of Xiao et al. The final aspect of the microplate, absorbance spectra, and illustrative calibration curve of different soluble starch standards (from left to right, respectively) of the starch-iodine quantitative assay used for the characterization of alpha-amylase and amyloglucosidase activities. where AVGmax is the average of the maximum absorbance values, SDmax is the standard deviation of the maximum absorbance values, AVGmin is the average of the minimum absorbance values, SDmin is the standard deviation of the minimum absorbance values, n is the number of tests performed. It is also crucial to accurately use well-defined conditions for laboratory transfer. [Crossref]. 7 0 obj Amylase is naturally produced in humans by the salivary glands and pancreas. 3. Slight changes in enzyme concentration, time duration prior to and during the incubation, and time in the water bath resulted in significant changes in the inhibition percentage. [Crossref]16 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.-1717 Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Curr. A state of sustained hyperglycemia caused by insulin deficiency can lead to type 2 diabetes.11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India, 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature, Aguiar, M.S., Maldonado, R.R., Carvalho, A.L., Aguiar-Oliveira, E. (2022). Dissolve 0.2M (2.7218 grams) of KH2PO4 in 100ml of distilled water to this solution add 0.5M (2.8053 grams) KOH drop by drop till the pH is set to 6.8. 2017, 74, 158. The -amylase assay was performed using Millers method, i.e., the DNS method. 29 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. The optimized and validated method was accurate, precise, and linear. The evaluation of the matrix effect showed linearity at 1 5 g mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Selected plant products could provide better alternatives due to their low cost and safer use. Tailorable assay based on the starch-iodine staining for the determination of alpha-amylase and amyloglucosidase activities. Some more thoughts: Try boiling the DNS solution longer than 5 min. Even though the water bath is at 100C, the internal solution temperature may no For measuring the amylase activity with DNS solution (Miller Method): 1. Prepare starch solution (10 mg/ml) 2. Prepare enzyme extract filtered with 5. The https:// ensures that you are connecting to the Rev. This evaluation revealed that minor modifications/changes in the assay conditions can lead to a significant variation in IC50 values (0.00068 to 47.5 mg mL-1) with acarbose used as a positive control. Estimation of Tyrosine by Folin-Ciocalteau Method, Estimation of Proteins by Lowry method (Quantitative Analysis). Med 2011, Article ID 810207. The development work herein reported sought to answer to the major methodological difficulties that we experienced for implementing the protocols for measuring enzyme activity (for both amylase and amyloglucosidase) and glucose yield (for amyloglucosidase), starting from the report of Xiao et al. official website and that any information you provide is encrypted We adopted this strategy to separate the solid particles that remained after the hydrolysis of different pure starches mediated by amyloglucosidase (glucose yield protocol, step 5). Then, we have partially purified these amylases employing three conventional methods: (a) cold acetone precipitation, (b) ion-exchange chromatography, and (c) gel filtration chromatography. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. Positive controls are compounds with proven activity in humans, whereas negative controls are those without activity. official website and that any information you provide is encrypted Acarbose was used as a standard for this activity. Please enable it to take advantage of the complete set of features! For the GOPOD method under microplate format, we also performed a similar method characterization. [Crossref], 10 White, R. E.; Annu. Med 2011, Article ID 810207. This is not surprising, considering the importance of tertiary structure in enzyme function and non-covalent forces in determining the shape of enzymes. The variability test is conducted at three response levels with varied signal intensities.1515 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. In this context, we herein describe an adaptation of a method proposed by Xiao et al. Universal buffer: o-phosphoric acid 47mmolL1, acetic acid 50mmolL1, boric acid 50mmolL1. [Crossref], 2 de Sales, P. M.; de Souza, P. M.; Dartora, M.; Resck, I. S.; Simeoni, L. A.; Fonseca-Bazzo, Y. M.; de Oliveira-Magalhes P.; Silveira, D.; Food Chem. Unable to load your collection due to an error, Unable to load your delegates due to an error. <>/Font<>/ExtGState<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI]>>/Contents[19 0 R 20 0 R]/Group<>/Type/Page/Tabs/S>> The site is secure. 14 Food and Drug Administration (USA); Bioanalytical method validation guidance for industry, US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research and Center for Veterinary Medicine, 2018, available at https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf, accessed June 2022. The experimental design and data handling were performed using Statgraphics Centurion XVI software (Statpoint Technologies, Inc., Warrenton, VA, USA). Amylase Substrate Mix 5 ml NM Amylase Positive Control (lyophilized) 1 vial Red Nitrophenol Standard (2 mM) 150 l Yellow Storage Conditions and Reagents Preparation Store kit at -20C. Dissolve 134.1g of malic acid, 70g of NaOH, and 58.4g of NaCl in 900mL of water (deionized or ultra-pure). I agree with all answers, try to raise the temperature reaction and be sure the pH buffer is ptimal for your amylase. In addition, the amylases re The structure of salivary amylase is a long, spiral-shaped molecule. Would you like email updates of new search results? For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. In excel file that you have prepared standard curve, ob <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>> The masses of soluble starch represented are 0.0, 0.75, 1.50, 2.25, and 3.00mg. Salivary amylase is produced by the salivary glands and released into the mouth when we eat. After 10min, the hydrolysis was stopped with the addition of 750L of 1molL1 HCl. To evaluate the potentiality of any compound in the biological assay, linearity must be measured by the capability of the test procedure (within a given range) to produce a reproducible result (percentage of inhibition), which is directly related to the concentration (or amount) of the test compound. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. To evaluate linearity, amylase inhibition studies were performed using different concentrations of the inhibitor acarbose (0.5 to 12 g mL-1). Amylase is an enzyme, or special protein, that helps you digest carbohydrates. Accessibility The proposed method showed high precision in long-term use (RSD<6.3%). Experiments conducted to evaluate plate uniformity and signal variability indicated that the response is not influenced by its location on the plate. The authors also acknowledge to Slvia Azevedo for the valuable technical assistance. A uniformity assay is performed at maximum and minimum response levels, ensuring that the difference is adequate to detect active compounds or the influence of the inhibitor in the assay system. A total of 64 experiments were performed. Standard deviation was calculated, and the relative standard deviation in terms of percentage was determined (Equation 2) to check the precision values on the experimentation day and other different days. Km was found to be 0.38 mg mL-1. Abstract:NelsonSomogyi and 3,5-dinitrosalicylic acid (DNS) assays are the classical analytical methods for the determination of activity of starch-debranching enzymes, however, they have a narrow detection range and do not adapt to the quantitative measurement of linear polysaccharides. 2015 Apr 22;164:210-22. doi: 10.1016/j.jep.2015.02.018. For each experiment, fresh and independent reagents were used, and the evaluation was performed on three different days. This enzyme is responsible for breaking down complex carbohydrates such as starch into simpler sugars such as maltose. Sci. The activity of enzymes is strongly affected by changes in pH and temperature. Amylase is an enzyme made by your pancreas and salivary glands that helps your body break down carbohydrates. By intercepting this absorbance values in the calibration curve (e.g. The linear range of the measurements included masses of soluble starch up to 3.00mg (see, The amount of hydrolyzed starch was the difference between the masses calculated from the calibration curve obtained by plotting absorbance. %PDF-1.5 Read the entire protocol before performing the assay. Your email address will not be published. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. [Crossref], 24 Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Ind. 1979 Nov;17(11):705-8. 6 This method, like the iodine procedure only measures ~-amylase activity and the results cannot be readily expressed in international units. Spectrosc. It then breaks down carbohydrates in the food we eat into simpler sugars. National Library of Medicine Acta Part A-Mol. https://www.americanlaboratory.com/913-T https://www.fda.gov/files/drugs/publishe https://www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf. Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. An amylase test measures the amount of amylase in your blood or urine (pee). It is a reversible, competitive inhibitor. It can also be naturally found in plants and animals. Pharmacol. A small amount of amylase in your blood and urine is normal. This indicates that the plant extracts contain a strong inhibitor of the amylase enzyme. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. There are three basic types of amylase: alpha, beta, and gamma. Furthermore, this improved method is very flexible and allows a broadband application, being able to be easily adjusted according to the requirements of each study or the equipment and instrumentation available at each laboratory. Some examples are reported in our previous publications [7,8]. Hi, Here I suggest a brief protocol: 1. You have to carry out the hydrolysis of the substrate (starch, I guess) by contacting a suitable dilution o However, the use of inhibitors is associated with certain side effects, such as diarrhea and other intestinal disturbances.11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. 2000, 40, 133. This method is based on the binding of iodine to terminals of the starchs polymeric chain [4] that results in a blue colored complex that can be also quantitatively monitored by UVvis spectrophotometry [5]. Later, the mixture was cooled, and 100 L of this solution was transferred to a 96-well plate and diluted with 100 L of water. Federal government websites often end in .gov or .mil. 2021 Nov 16;10(11):2818. doi: 10.3390/foods10112818. Statistical analysis and validation were performed using Microsoft Excel 2010. Here we adopted the same detection wavelength of the original method (580nm) [5], which is associated with an amylopectin/amylose ratio of 80:20 [12]. 2000, 40, 133. To determine activity of Amylase enzyme in Saliva. Those with normal plasma glucose had group I, those with impaired glucose tolerance had group II, and those who were diabetic had group III. Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. Epub 2020 Oct 26. <>>> All subjects were subjected to fasting blood glucose estimation followed by measurement of plasma glucose level after 75g of glucose ingestion. 2011, 44, 2076. Determination of kinetic constants was performed by determining the effect of substrate concentration on enzymatic activity. In a tube containing 1 mL of phosphate buffer (100 mmol L-1, pH 6.9), 100 L of -amylase (final concentration 0.15 U mL-1 in phosphate buffer) was added and incubated at 37 C for 7.2 min for preincubation. One negative control (absence of inhibitor) was set up to obtain 100% enzyme activity. 2011, 136, 305. When compared with the use of soluble starch, we adjusted the volume of HCl for stopping the reaction (200L instead of 750L) in order to maintain the buffering capacity of the GOPOD reagent (glucose yield protocol, step 4). It has long been known that salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus. The authors are also grateful for the financial support of the Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES, Brazil) under grant number 451408/2019-01 and the Fundao de Amparo Pesquisa do Estado da Bahia (FAPESB, Brazil) under grant number RED0023/2014. 2012, 13, 427. In the case of using a particular starch as a substrate, this factor should be studied in detail and the method should be adjusted accordingly. The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. Achillea fragrantissima Forssk. Evaluation of precision and accuracy was performed using the extracted sample of J. dioica in the presence or absence of acarbose (positive control). Jarvis C.E., Walker J.R.L. - 204.93.178.112. Chem. The Millers reactions were performed in 13 100 test tubes. In most of these applications, starch is submitted to a hydrolysis process that aims the conversion of the polymeric chain composed by two high-molecular weight units (amylose and amylopectin) into its primary units (glucose) [2] mediated by amylolytic enzymes. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. The residues used correspond to the fruit peels and the pressed cake obtained after extracting the vegetable oil from its seeds. The enzyme concentration, incubation time, interaction between enzyme concentration and preincubation time, and the interaction between preincubation and boiling time showed a significant effect on the inhibition percentage. Epub 2018 Sep 19. It is then important to control the activity of these enzymes in two major complementary roles: as a quality control tool, for monitoring enzyme activity along time and batch to batch reproducibility in different industrial and laboratory applications, and also as a research tool, for studying novel strategies to improve the efficiency of the overall process. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Insoluble chromogenic substrates have been developed for ~-amylase assay. 19 0 obj The amount of Maltose in the given unknown sample is _________ grams of Maltose formed per 100ml of enzyme per one hour. The solution with varying concentrations of acarbose was placed in 32 wells in three different plates. Some of them are essential for the operation of the site, while others help us to improve this site and the user experience (tracking cookies). Acarbose, a positive control, was used as an inhibitor for this experiment. These bonds can be easily broken by water, so saliva is necessary for the enzyme to work. Determination of amylose/amylopectin ratio of starches. For the starch-iodine method, absorbance measurements were conducted at 580nm, whereas for the GOPOD assay, the wavelength was set at 505nm. 2019 Oct;18(4):2531-2539. doi: 10.3892/etm.2019.7840. Insights into the Inhibition Mechanism of Human Pancreatic -Amylase, a Type 2 Diabetes Target, by Dehydrodieugenol B Isolated from Ocimum tenuiflorum. LOD was calculated as the starch mass equivalent to the absorbance that resulted from the addition of the blank signal (standard solution without soluble starch) to 3 times the corresponding standard deviation [14]. The absorbance of the product was recorded at 540 nm. Constructing a standard curve / graph for maltose helps us to estimate concentration of reducing sugars present in an unknown sample and for determining the activity of amylase enzyme in Int. The preliminary dissolution in cold water, followed by the dissolution in hot water, and then the mixing and cool down (maintaining the stirring) are critical aspects to obtain a clear (and not cloudy) solution of soluble starch. Cool and dilute with 10ml of distilled water. J Clin Chem Clin Biochem. Amylase is also used in brewing and baking, as it helps to convert starch into sugar. The DNS method comprises a complex and labor-intensive protocol that includes heating and the use of potentially harmful reagents (e.g. Salivary amylase is the enzyme produced by the salivary glands. 2012, 13, 427. Correspondence to MeSH It was initially termed diastaste but was later renamed amylase in the early 20th Hence, we kept the same volume ratio of the original method but we increased the assay to a total volume of 3.00mL according to our requirements, which were defined by the specifications of the US probe used to study the effect of US in the activity of amylolytic enzymes [7,8]. Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. The relative standard deviation and Z value were found to be <4% and >0.93, respectively. [Crossref], 31 Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Molecules 2022, 27, 774. J Genet Eng Biotechnol 14:133141, CrossRef This process starts in the mouth and continues in the stomach and intestines. endobj [5] for measuring the activity of alpha-amylase and amyloglucosidase using the starch-iodine assay principles. Acarbose and miglitol are conventional inhibitors used to inhibit the activity of enzymes. 4 FOIA It was experimentally established that the minimum concentration of acarbose that can be determined is 1 g mL-1. These extractions are commonly performed with buffer or salt solutions at mild temperatures (from room temperature to 40C). Absorbance is increasing with increasing concentrations of your inhibitor? 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