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Poster Sessions: Chemistry

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Title:
Poster Sessions: Chemistry
Series Title:
Quest
Creator:
Bailine, Ryan
Boafo, Clara
Brown, Thomas
Burley, Kyle
Burrell, Tanashia
Kearns, Ryan
Kincaid, Warren
Koeppe, Julie
Kuhn, Derek
Pokharel, Namrata
Punzalan, Mikaya
Raymond, Brooke
Roque, Johann
Publication Date:
Copyright Date:
2021

Notes

Abstract:
Determination of Pb in orchard soil samples at the Rice Creek Field Station by Warren Kincaid. Levels of lead (Pb) in former orchard soils were sampled to determine lead contamination at varying depths below the ground surface. Four sites of Rice Creek Field Station’s abandoned orchard outside of Oswego, NY were sampled with their geographic locations identified. Samples were measured and divided consistently by their depth below the soil surface. Samples were then dried, ground, ashed, and digested for analysis using atomic absorption spectroscopy. Concentrated nitric acid, HNO3, was added to digest and elute the lead from the soil into solution. Use of the insecticide lead arsenate, by orchard farmers of the early and mid1900s-, is likely a main source of this lead being unearthed. Lead typically resides in sandy soils of the O and A horizons. The importance of this investigation into lead contamination is the possible lead exposure to those who visit this now protected nature area as well as to the residents of the surrounding Rice Creek nature area. ( ,,,,,,, )
Abstract:
Analysis of Degradation Pathways for a Biodegradable Plastic by Kyle Burley and Johann Roque. Stimuli-responsive plastics are materials that are capable of responding to their environment to change their physical properties. These dynamic materials have seen significant growth over the past decade due to their versatility. One field of stimuli-responsive plastics that is of particular interest is degradable plastics. Globally we are facing a plastic waste issue that becomes more detrimental each year. In an effort to develop environmentally-friendly plastics, this research group has developed a stimuli-responsive plastic capable of responding to UV-light, leading to degradation. However, the particular pathway of degradation has yet to be determined. This project focuses specifically on the synthesis of molecular model systems that can be used to determine how our plastic is breaking down in response to light.
Abstract:
Investigating the function of enzyme 2O14 by Ryan Bailine and Derek Kuhn. There are over 3800 structures of unknown function available in the Protein Data Bank (PDB). Of these, we have been working to characterize the enzyme with PDB ID 2O14. Computational analysis indicates that this enzyme is an esterase or lipase, which will hydrolyze ester bonds. We expressed the enzyme in E. coli before isolating the cells by centrifugation and using sonication to break the cell membrane and release the enzyme of interest. We passed the collected supernatant through a Ni-NTA affinity chromatography column to isolate the His-tagged 2O14. We identified which fractions contained 2O14 by using gel electrophoresis (SDS-PAGE) to analyze samples. We then performed a Bradford assay to determine the concentration of the enzyme. To test for the predicted esterase or lipase function, we carried out enzymatic assays using chromogenic substrates. Both the ester substrate, p-nitrophenyl acetate (PNPA), and the lipid substrate, p-nitrophenyl dodecanoate, produce a yellow-colored product when hydrolyzed. In order to test the lipid substrate, we must include detergent in the assay buffer. For this reason, we are also testing hydrolysis of the PNPA substrate in the presence and absence of detergent to understand the effect of the detergent on the enzymatic activity of 2O14.
Abstract:
Quantitative Analysis of Diphenhydramine Using Dispersive Liquid-Liquid Microexctraction (DLLME) Paired with GC-MS by Ryan Kearns. Drug-facilitated crimes (DFC) have been an age-old practice, however over the past three decades the prevalence of DFC has increased to the point of public concern. Recently, a common over-the counter drug diphenhydramine (DPH) has become commonly used as a date rape drug. DPH is highly potent with a short half-life making detection difficult abruptly after a drugging occurs. This work is concerned with the refinement of a dispersive liquid-liquid microextraction (DLLME) method of DPH and other antihistamines from aqueous solution. Gas chromatography coupled with mass spectroscopy (GC-MS) is used for analysis in order to detect DPH in parts per billion (ppb) concentrations. Aqueous solutions of DPH were prepared in a range between 0.005-2 mg/L. Buffered aqueous solutions were spiked using 1mL of a 13:40 ratio of a mixture of acetone and toluene used as dispersive solvent and extraction solvent, respectively. Utilizing liquid nitrogen, the aqueous layer was frozen, and the toluene layer was collected. To increase extraction recovery, a second extraction was performed. The collected toluene layers were evaporated and reconstituted using MeOH, then analyzed by GC-MS. Standard calibration solutions of DPH were prepared and analyzed by GC-MS, and extraction recoveries were calculated.
Abstract:
Prediction of Function for Enzyme 4Q7Q by Tanashia Burrell and Mikaya Punzalan. The goal of our project is to determine the function of an enzyme for which the three-dimensional structure is already known. According to the Protein Data Bank (PDB), 4Q7Q is the crystal structure of a possible lipase from Chitinophaga pinensis. Computational sequence and structure alignments were used to predict the function and determine where the active site is in the 4Q7Q structure. We then expressed this enzyme in E. coli before isolating the cells by centrifugation and using sonication to break the cell membranes and release the enzyme of interest. We passed the collected supernatant through a Ni-NTA affinity chromatography column to isolate the His-tagged 4Q7Q. We used gel electrophoresis (SDS-PAGE) to analyze samples from the column in order to determine which of these contained our target enzyme. The Bradford assay technique was usedAnalysis of to determine the concentration of the enzyme 4Q7Q. Chromogenic substrates were used for the enzymatic assays so that the function of esterase or lipase could be determined. We have demonstrated that 4Q7Q can hydrolyze p-nitrophenyl acetate, which supports its function as an esterase. We are using two different lipid substrates, p-nitrophenyl decanoate and p-nitrophenyl dodecanoate to test for lipase function. We are comparing the activity of 4Q7Q as a lipase using either bile salts or phospholipid vesicles to solubilize the lipid substrates.
Abstract:
Analysis of Processed Oats by Using Infrared Analysis by Clara Boafo. The objective of the project was to find out the variability in food processing by measuring moisture, test weight, outliers, and protein DM in oats. The oats stated above was tested to assess if protein DM strays from one another. The results gained from the GAC 2500 UGMA Machine showed that the test weight all fell within the range of 10-13%, the test weight of 29, 30 and 57 lbs/bu. According to the Infratec Analysis Machine the protein DM was around 14% and the moisture was approximately 10%.
Abstract:
Cortisol Measurements in Animals by Brooke Raymond. Cortisol levels have been studied in various species to understand chronic stress and other health-related factors in domestic and wild animals. Samples such as blood, urine, feces, hair, and nails have been collected from animals to quantify their cortisol levels. Some of these samples have proven difficult to obtain and some cause further stress on the animal. In a collaborative project with the Veterinary College at Long Island University, cortisol levels were quantified in 35 domestic animals, where two different samples in each animal were collected (N=70). Samples were mechanically disrupted, and cortisol was extracted from each. An overnight in methanol extraction was followed up by a 5-minute acetone wash. The process was repeated over two more nights to increase yields. Samples were dried under nitrogen, dissolved in a buffer, and cortisol was quantified using an antibody-based assay called ELISA. The results showed a difference between the cortisol levels in the two types of samples collected. Applications of these sample collection and extraction methods in veterinary medicine could lead to a better understanding of chronic stress and other health-related factors in domestic animals.
Abstract:
Testing for esterase function in the enzyme with PDB ID 4Q7Q by Namrata Pokharel. Enzymes are vital for life and all of life can be explained and better understood through enzymatic studies. The purpose of this project is to study and predict the function of an enzyme by using computational analysis and enzyme kinetics. Among the numerous enzymes of unknown function the enzyme we are working with is a possible lipase from Chitinophaga pinensis DSM 2588 with the PDB ID 4Q7Q. We have expressed this enzyme in E.coli and purified it with the help of Ni-NTA affinity chromatography. Since protein function is often tied to structure, structural comparisons and studies were done using computational tools. Lipase function was predicted after performing both global and local structure alignments using Dali and ProMOL. This function was supported by sequence alignment data from BLAST and PFam. To test for esterase function, enzymatic assays were carried out using two short chained substrates, p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB). We will determine whether the enzyme has a preference for a longer or a shorter chain ester-containing molecule by comparing these results to hydrolysis of lipid-like substrates with longer carbon chains. We are in the initial stages of these tests and we hope to duplicate our first set of results which show significant esterase activity with the p-nitrophenyl acetate substrate.
Summary:
Session Chair: Julia Koeppe
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Collected for SUNY Oswego Institutional Repository by the online self-submittal tool. Submitted by Zach Vickery.

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Legend Blue: 0 mg/mL 4Q7Q Orange: 0.05 mg/mL 4Q7Q Yellow: 0.1 mg/mL 4Q7Q Grey: 0.2 mg/mL 4Q7QFigure 4 : Kinetic analysis of 4 Q 7 Q . Varying concentrations of 4 Q 7 Q were tested to measure hydrolysis activity with two different substrates . The substrate concentration was the same for all trials, and 4 Q 7 Q showed hydrolysis that was dependent on enzyme concentration with both substrates. A B C AbstractThe goal of our project is to determine the function of an enzyme for which the three-dimensional structure is already known. According to the Protein Data Bank (PDB), 4 Q 7 Q is the crystal structure of a possible lipase from Chitinophaga pinensis . Computational sequence and structure alignments were used to predict the function and determine where the active site is in the 4 Q 7 Q structure . We then expressed this enzyme in E . coli before isolating the cells by centrifugation and using sonication to break the cell membranes and release the enzyme of interest . We passed the collected supernatant through a Ni -NTA affinity chromatography column to isolate the His-tagged 4 Q 7 Q . We used gel electrophoresis (SDS PAGE) to analyze samples from the column in order to determine which of these contained our target enzyme. The Bradford assay technique was used to determine the concentration of the enzyme 4 Q 7 Q . Chromogenic substrates were used for the enzymatic assays so that the function of esterase or lipase could be determined . We have demonstrated that 4 Q 7 Q can hydrolyze p -nitrophenyl acetate, which supports its function as an esterase. We are using two different lipid substrates, p nitrophenyl decanoate and p nitrophenyl dodecanoate to test for lipase function. We are comparing the activity of 4 Q 7 Q as a lipase using either bile salts or phospholipid vesicles to solubilize the lipid substrates . Introduction Purification and Analysis of 4Q7Q Testing 4Q7Q Activity Future Work References AcknowledgementsThe Investigation of Structure & Function for the Enzyme 4Q7QTanashia Burrell, Mikaya Punzalan, and Dr. Julia KoeppeDepartment of Chemistry, State University of New York at OswegoAccording to the Protein Data Bank (PDB), the enzyme 4 Q 7 Q contains two chains denoted with the letters A and B in its structure, and it is proposed to be a lipase . The structure was determined at the Midwest Center for Structural Genomics . We performed sequence and structure alignments using BLAST, Pfam , Dali, and Moltimate . Each alignment suggested that 4 Q 7 Q is an esterase or lipase . Figure 2 : SDS PAGE analysis of fractions from NiNTA chromatography . The samples that were loaded into the wells are 1 ) molecular weight marker 2 ) cleared lysate 3 ) column flow through 4 ) wash 1 5 ) wash 2 , and 6 10 ) column elution fractions 1 5 . Figure 1 : The structure and proposed active site of 4 Q 7 Q . (A) 3 D structure of the A chain of 4 Q 7 Q . (B) Alignment of Gly 69 , Asn 97 , Asp 29 , Ser 30 , Asp 251 , and His 254 from 4 Q 7 Q (green) with the active site of 1 BWP (blue), an acetylhydrolase . This alignment has an RMSD of 0 . 4725 . (C) Proposed active site of 4 Q 7 Q shown in red on the A chain of the 4 Q 7 Q homodimer (figure prepared using PyMOL ) . [ 1 3 ] Figure 3 : Bradford assay standard curve used to determine 4 Q 7 Q concentration . From the linear fit of the standards, we determined that the concentration of 4 Q 7 Q was 0 . 659 mg/ mL .After completing the computational analysis, we prepared a sample of 4Q 7Q in the lab in order to complete testing of the proposed lipase activity . A plasmid containing the gene for 4 Q 7Q was used to transform E . coli BL 21(DE 3 ) cells . We then grew transformed cells in autoinduction medium to produce the 4 Q 7 Q enzyme . After collecting the cells and lysing them by sonication, we purified the 4Q 7Q using NiNTA chromatography in which the His tagged 4Q 7 Q bound to the column while other proteins flowed through. We used SDSPAGE and a Bradford assay to analyze our purified sample .Going forward with the investigation of structure and function of 4 Q 7 Q we plan to test for activity with varying lipid substrate and constant enzyme concentration . This will be done by using 0 . 05 mg/mL 4 Q 7 Q enzyme concentration with varying p nitrophenyl dodecanoate from 0 mM up to saturating conditions (initial trials were 0 1 . 5 mM substrate concentration) . Likewise, we will also be testing another lipid substrate, p nitrophenyl decanoate. We hope to determine Michaelis Menten kinetic parameters with other students who are testing ester substrates ( p nitrophenyl acetate and p nitrophenyl butyrate) . Because 4 Q 7 Q is predicted to be an esterase or lipase, we tested for enzyme activity using two chromogenic substrates . p Nitrophenyl acetate (PNPA) is a small ester substrate, and p nitrophenyl dodecanoate is a longer lipid substrate . Both substrates produce a yellow product, p nitrophenolate ion, when hydrolyzed. Based on the linear fit of these graphs, the slope can be interpreted as the velocity of the reaction . This can be represented as or . •SUNY Oswego Department of Chemistry • Namrata Pokharel , Slater Bushen, and Christopher Serrano for assistance with production and purification of 4Q7Q. • Dr.Kestutis G. Bendinkas1. https : // www . rcsb . org 2. moltimate . org 3. The PyMOL Molecular Graphics System, Version 2 . 0 Schrdinger, LLC .



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Brooke Raymond and Kestutis Bendinskas Department of Chemistry, SUNY at Oswego Collaborator: Dr. Elena Contreras, DVM, MS, PhD Goals Goals Cortisol Measurements in Animals Cortisol Measurements in Animals Results Results Discussion Discussion References References Homeostasis is a complex dynamic equilibrium kept by living organisms . 1 Homeostasis must be kept in order for an organism to stay alive . This equilibrium is influenced by stressors . A stress response occurs when homeostasis is threatened, or when an animal perceives that there is a potential threat to them . When an animal becomes stressed, the hypothalamic pituitary adrenal axis (HPAA) is activated . 2 Glucocorticoids are the final outputs of the HPAA, with cortisol as the main output in mammals . 2 By examining cortisol levels, it can be determined what factors influence acute (short term) or chronic (long term) stress, and how it can affect the health of animals . Cortisol can be measured in biological samples, such as plasma, saliva, urine, feces, milk, hair, nails, and sweat . 2 Blood, saliva, sweat, and milk cortisol levels are used to determine acute stress, while cortisol in urine and feces accumulates over several hours . Hair and nail samples can provide a look at chronic stress or an past exposure of stress . Our method was found to be a reliable way to determine cortisol levels in animals . This presents a new way to determine cortisol levels in these domestic animals . These methods could be applied in veterinary medicine to diagnose and treat animals with various conditions . This could lead to a further understanding of chronic stress in animals . The purpose of this research was to determine the cortisol levels in 35 domestic animals . By doing this, we could determine whether these samples were a reliable way to test cortisol levels in animals . We also wanted to compare two different conditions in order to see if there was a significant difference in cortisol levels between the two conditions . 1. Chrousos, G. P. Stress and Disorders of the Stress System. Nature Reviews Endocrinology 2009 , 5 (7), 374 381. 2. Casal, N.; Manteca, X.; Peña L, R.; Bassols, A.; Fàbrega, E. Analysis of Cortisol in Hair Samples as an Indicator of Stress in Pigs. Journal of Veterinary Behavior 2017 , 19 , 1 6. 3. Armbruster, D. A.; Pry, T. Limit of Blank, Limit of Detection and Limit of Quantitation . Clin Biochem Rev 2008 , 29 (Suppl 1), S49 S52. Acknowledgments Acknowledgments We thank our collaborator Elena Contreras at the Long Island University College of Veterinary Medicine and Charlotte Labrie Cleary of SUNY Oswego . Introduction Introduction Standard Curve Standard Curve Experimental Experimental A 1000 µL portion of methanol was added to the samples, and the samples were vortexed . The samples were then incubated in a 55 ° C oven overnight . The methanol portion was removed from the samples and put into another vial, and a 1000 µL portion of acetone was added to the samples . The samples were incubated at room temperature for five minutes . The portion of acetone was removed and stored in the supernatant vial . Another portion of methanol was added to the sample and again was incubated at 55 ° C overnight . There was a total of three overnight extractions . The supernatant was then thoroughly dried and stored at 4 ° C . Enzyme immunosorbent linked assay (ELISA) : A 1000 µL portion of acetone was added to the dried supernatant, then dried again . A 150 µL portion of assay dilutant was added, then heated in a 55 ° C water bath water for 5 minutes and centrifuged at 4000 rpm for 15 minutes . The sample was then pipetted into the ELISA plate . A high sensitivity salivary cortisol enzyme immunoassay kit was used to analyze the samples in duplicate . The absorbance was read at 450 nm . 0 0.5 1 1.5 2 2.5 3 3.5 0.01 0.1 1 10 Absorbance Concentration ( g/dL) Standard Curve showing the Experimental vs. Fitted Data Fitted Experimental The average cortisol concentration found in condition 1 was 1 . 938 pg /mg, while the average concentration in condition 2 was 2 . 617 pg /mg . The average concentration of cortisol in condition 2 was significantly higher than that found in condition 1 (t 35 = 2 . 26 , P = 0 . 027 ) . One sample was determined to be an outlier and was not included in the analysis . The limit of the blank (LOB) 3 was found to be 0 . 0151 microg/dL . One sample was found to be below the LOB . The limit of detection (LOD) 3 was found to be 0 . 0206 microg/dL . Three samples were found below the LOD . Sample Preparation Sample Preparation The samples were collected by Dr . Contreras and her team at Long Island University . The samples were washed four times with isopropanol in order to rid of any external contamination . The samples were thoroughly dried in a 55 ° C water bath under a stream of nitrogen . The samples were then weighed for future analysis .



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Analysis of Degradation Pathways for a Biodegradable Plastic Kyle Burley 1 , Johann Roque 1 , and Dr. Matthew Baker 1 1 Department of Chemistry, State University of New York at Oswego, Oswego, NY Introduction : Degradable plastics (also known as polymers) have become an increasingly important focus in plastics research . Over the past few decades, scientists and society as a whole have become aware of the plastic waste issue developing globally . In the United States alone, almost 400 million tons of plastic are being produced each year, with 79 % percent of that plastic ending up in the environment or landfills . 1 As a result, scientists are looking for ways to mitigate the long term impacts of plastic on the environment . One approach for alleviating the plastic waste issue focuses on developing plastics with the ability to break down using external stimuli . A stimulus of particular interest for this task is UV light due to the control scientists have over it . 2 Light can be easily introduced and removed from a system . In addition, multiple variables can be changed, such as the wavelength of the light and the intensity of the light . Herein, we describe the analysis of a photodegradable plastic developed in our lab . Our goal: The goal of this project was to identify how this degradation occurs . Degradation of plastics generally occurs through one of two different paths, randomly throughout the chain or selectively from one end of the polymer to the other . In order to test the degradation of the polymer, we synthesized two control molecules, one that represents the structure at the end of the chain ( 4 ) and one that reflects the structure within the chain ( 6 ) . Once those molecules have been synthesized, we will expose them to UV light to determine which, or if both, degrade . Figure 1 . Image showing degradation of poly( hydroxycinnamic acid) ( 1 ) to form coumarin ( 2 ) when exposed to UV light . Conclusion : The synthesis of both control molecules was successful . We will now turn our attention to the degradation of these molecules upon exposure to UV light and characterizing the outcomes . Synthesis: To create the control molecules, we converted a carboxylic acid to an acid chloride using thionyl chloride . Subsequently, phenol was added, completing the synthesis . For the control molecule representing the chain end, a free aryl alcohol is present (scheme 1 a), and for the internal control molecule, the phenol is protected with an acetate group (figure 1 b) . References: 1) Geyer, R.; Jambeck , J. R.; Law, K. L. Scientific Advances, 2017, 3, e1700782. 2) J . Olejniczak , C. J. Carling, and A. Almutairi , J. Control. Release, 2015, 219, 18 30; A. M. Kloxin , M. W. Tibbitt, A. M. Kasko , J. A. Fairbairn, and K. S. Anset , Adv. Mater. 2010, 22, 61 66. Scheme 1 . Synthesis of poly( hydroxycinnamic acid) Figure 3. Proton NMR spectra showing the formation of our two control molecules, the free phenol and the protected phenol respectively. Previous work : To date, significant progress has been made on this project . Previous students have been able to synthesize the desired stimuli responsive plastic, poly( 2 hydroxycinnamic acid) ( 1 ) from 2 hydroxycinnamic acid . Subsequently, they were also able to prove that the polymer was able to undergo degradation, forming coumarin ( 2 ) upon exposure to UV light . C omplete degradation of the stimuli responsive plastic was demonstrated using a gel permeation chromatography (GPC) instrument . We were able to show that the polymer completely degraded over the period of 96 hours, producing coumarin as the product . Results: Once the desired control molecules were synthesized, they were purified via column chromatography and characterized using nuclear magnetic resonance (NMR) imaging, as shown in figure 3 . According to proton NMR, we were able to synthesize control molecules 4 and 6 .



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Quantitative Analysis of Diphenhydramine (DPH) Using Dispersive Liquid Liquid Microextraction (DLLME) Paired with Gas Chromatography Mass Spectrometry (GC MS) Ryan Kearns , Shokouh Haddadi Department of Chemistry, SUNY Oswego, Oswego, NY 13126 Introduction Purpose Methods Results Conclusion Acknowledgements The authors would like to thank SUNY Oswego Chemistry Department and SUNY Oswego SCAC grant for providing financial support. Special thanks to Dr. Vadoud Niri, Dr. Matthew Baker, Kristin Gublo , and Elizabeth Brown. Diphenhydramine (DPH) is a peripheral H 1 receptor antagonist drug with antihistaminic properties . Benadryl is a common over the counter antihistamine, which contains DPH as active ingredient . DPH possesses sedative properties and has been reported as being used in drug facilitated crimes (DFC) and has been classified as a rape (Carter J . et al . 2000 ) . Many perpetrators have been using DPH to spike alcoholic drinks at bars and clubs due to its synergistic properties with alcohol and ease of access . Normally, sedation/drowsiness occurs after a concentration of 30 40 ng/mL is within the blood stream ( 1 2 pills) and mental impairment at 60 ng/mL ( 2 + pills) (Couper F . et . al) . However, when paired with the sedative affects of alcohol it takes a smaller dosage for these symptoms to occur due to their synergism . DPH has a short half life of 4 8 hours making it difficult to detect based on the already low concentration of its initial dosage being rapidly metabolized . This is a common challenge in forensic toxicology with improved/cost effective methods being sought after for the detection and quantification of various drugs and compounds such as DPH . Results Continued The results suggest that the DLLME procedure is successful in the extraction of DPH from aqueous solutions based on the retention times found for the standard and aqueous samples . Based on the extraction recoveries, a double extraction may not prove necessary ; however, it seems to have better reproducibility compared to a single extraction and does not take too long to perform . The results were promising in the success of this project, but further work is needed with the use of synthetic urine samples and the creation of a calibration graph . Also, GC MS should be used to achieve lower detection limits . Methods Continued References Aqueous Sample Spiking with Dispersive Solvent Dispersion of Solvent Separation of layers Collection Figure 1: D iagram of DLLME procedure. Figure 3 : Comparison of extraction recoveries with standard deviation error Future Goals Prepare aqueous solutions containing DPH in a 5 ng/mL (ppb) to 2 (ppm) range, and perform a double extraction using DLLME to test detection at ppb levels . Prepare direct calibration solutions containing DPH in a 250 ng/mL (ppb)to 100 (ppm) range and run all samples in GC FID or GC MS for analysis . Explore the extraction of a mixture of antihistamines (DPH, Cetirizine, and Hydroxyzine) using DLLME . Instrumental analysis Thermo Scientific Trace 1310 GC Injection temperature: 250 Temperature program: Held at 1 for 9 min Increased to 250 at 25 /min Held for 2 min at 250 Total run time: 15 min Detector temperature: 300 The goal of this work is to develop a dispersive liquid liquid microextraction (DLLME) method to exhaustively extract DPH from aqueous solutions before separation, detection, identification, and quantification by gas chromatography coupled to mass spectrometry (GC MS). The method was first tested with gas chromatography with flame ionization detector (GC FID). The proposed and tested DLLME method for extraction of DPH from standard aqueous solutions of DPH: Preparing Aqueous DPH Sample (5 ppm): 50 µ L of n Butanol was added around the glass of six 15 mL glass centrifuge vials using a micropipette. 5 mL Ultra Pure water was pipetted into the centrifuge vials. 25 µ L of 1000 ppm stock DPH added to vials using a micropipette. Samples were sonicated for 15 minutes and stored at 4 inside a fridge until extractions were performed. Preparing Standard Methanolic Solution (250 ppm): 75 µ L of methanol was transferred into 2 mL brown stained glass vial using a micropipette. 25 µ L 1000 ppm DPH stock solution was added to the vial using a micropipette. The solution was sonicated and stored at 4 inside a fridge until GC analysis was performed. DLLME Extraction of DPH from Aqueous Samples: 20 µ L of phosphate buffer and 50 µ L of 2 M NaOH were added to each aqueous solution to adjust the pH at 12. Samples were shaken for 10 minutes and left to settle. Samples were then spiked with 1 mL of DLLME solvent mixture (toluene: acetonitrile, 13:40), and centrifuged for 10 minutes. Samples were then placed into an ethyl acetate bath in a holding tray and liquid nitrogen was added to freeze the aqueous layer (5 minutes). Unfrozen toluene layer (top layer) was collected form the samples using a micropipette and added into a 2 mL brown stained glass vial. A double extraction was performed on three of the six aqueous samples. Evaporation and Reconstitution of Aqueous Samples: The 2 mL brown stained glass vials containing the collected toluene layer were connected to a Schlenk line in order to evaporate the solvent. Using nitrogen gas, the vials were dried after evaporation. Reconstitution was performed by adding 100 µ L methanol around the inside of the vial. The samples were sonicated and allowed to homogenized at 4 Figure 2: Freezing the aqueous layer in ethyl acetate bath and evaporation of toluene. Utilizing GC FID the extraction recoveries were calculated using the following equation: Table 1 . Extraction recoveries of single and double extraction J. Carter. (1998, July 27) Controlled and Uncontrolled Substances Used to Commit Date Rap e (2000) Controlled and Uncontrolled Substances Used to Commit Date Rape (house.gov) . Couper F, Logan B. NHTSA Drugs and Human Performance Fact Sheet (2004, April) https://mn.gov/law library stat/archive/urlarchive/a080579 1.pdf 0 20 40 60 80 100 120 Single Extraction Double Extration Extraction Recoveries Identification of DPH The identification of the extraction of DPH was confirmed by the retention times in Table 1. The comparison of the effects of a double extraction on extraction recovery can be seen in Figure 3. Sample Retention Time (min) Peak Area Extraction Recovery Average Extraction Recovery Standard Deviation (s) %RSD 5 ppm ( aq )(1) 13.192 0.6574 105 92 18 19 5 ppm ( aq )(2) 13.192 0.4534 72 5 ppm ( aq ) (3) 13.196 0.6270 100 5ppm 2x ( aq ) (1) 13.194 0.5381 86 96 11 11 5ppm 2x ( aq ) (2) 13.196 0.6741 108 5ppm 2x ( aq ) (3) 13.196 0.5979 95 Direct 250 ppm 13.196 0.6266



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ResultsKinetic Analysis: The initial kinetic testing has revealed that 4 Q 7 Q hydrolyzes both the pnitrophenyl acetate and pnitrophenyl butyrate substrates . These substrates both produce a yellow product, pnitrophenolate ion, when hydrolyzed, so the reactions can be followed by monitoring the absorbance at 405 nm (Figure 6 ). Trials were carried out to determine what quantity of enzyme was needed to demonstrate hydrolysis . Following this, the 4 Q 7 Q concentration was kept at 0 . 05 mg/mL while the substrate concentration was varied in order to establish whether 4 Q7 Q follows the Michaelis Menten kinetic model and attains saturation at high concentrations of enzyme .Further TestingThe initial testing has shown promising results but the immediate next step will be replicating the results for consistency . Once that is completed, the Michaelis Menten parameters of Vmaxand KMcan be determined and compared for the different substrates . There are also further substrates to be tested including longer chain substrates like pnitrophenyl decanoate and pnitrophenyl dodecanoate. Since computational analysis showed similarities in the sequence of 4 Q7 Q with known lipolytic proteins lipid substrate testing is also going to be another goal.References1. The Protein Data Bank H. M . Berman, J . Westbrook, Z . Feng, G . Gilliland, T . N . Bhat, H. Weissig , I . N . Shindyalov , P . E . Bourne ( 2000) Nucleic Acids Research , 28: 235242 . doi : 10 . 1093 / nar / 28 . 1 . 235 2. Moltimate . org 3. The PyMOL Molecular Graphics System, Version 2 . 0 Schrdinger, LLC . 4. Madden T . The BLAST Sequence Analysis Tool . ( 2002) 5. Holm L ( 2020) DALI and the persistence of protein shape . Protein Science 29, 128140. 6. The Pfam protein families database in 2019 S . El Gebali, J . Mistry, A . Bateman, S . R . Eddy, A . Luciani , S . C. Potter, M . Qureshi, L . J . Richardson, G . A . Salazar, A . Smart, E . L . L . Sonnhammer , L . Hirsh, L . Paladin, D . Piovesan , S . C. E . Tosatto , R . D . Finn ( 2019) Nucleic Acids Research . doi : 10. 1093 / nar /gky 995 7. Grosdidier A , Zoete V, Michielin O . SwissDock , a protein small molecule docking web service based on EADock DSS. ( 2011 ) Nucleic Acids Res . W 2707 . doi : 10. 1093/ nar /gkr 366 . 8. Lopes, Danielle Branta , Fraga , Laira Priscila, Fleuri, Luciana Francisco, & Macedo , Gabriela Alves . Lipase and esterase: to what extent can this classification be applied accurately? . ( 2011 ) Food Science and Technology , 31( 3 ), 603 613. https : //doi . org/ 10 . 1590 /S 0101 20612011000300009. AbstractEnzymes are vital for life and all of life can be explained and better understood through enzymatic studies . The purpose of this project is to study and predict the function of an enzyme by using computational analysis and enzyme kinetics. Among the numerous enzymes of unknown function the enzyme we are working with is a possible lipase from Chitinophaga pinensis DSM 2588 with the PDB ID 4 Q 7 Q . We have expressed this enzyme in E . coli and purified it with the help of Ni NTA affinity chromatography . Since protein function is often tied to structure, structural comparisons and studies were done using computational tools . Lipase function was predicted after performing both global and local structure alignments using Dali and Moltimate . This function was supported by sequence alignment data from BLAST and PFam . To test for esterase function, enzymatic assays were carried out using two short chained substrates, p nitrophenyl acetate (PNPA) and p nitrophenyl butyrate (PNPB) . We will determine whether the enzyme has a preference for a longer or a shorter chain ester containing molecule by comparing these results to hydrolysis of lipid like substrates with longer carbon chains . We have duplicated our first set of results which show significant esterase activity with the p nitrophenyl acetate substrate and p nitrophenyl butyrate . The next step would be to test longer chain substrates, lipid substrates and vesicles.IntroductionEnzymes are vital for life and all of life can be explained and better understood through enzymatic studies . They assist in metabolic processes, break large molecules into smaller pieces that are more easily absorbed by the body, and help bind two molecules together to produce a new molecule . Enzymes allow all reactions to occur at the rate necessary for life . Understanding how enzymes work and what unique function each enzyme has can help us make major headway in the study of evolution, medicine, and just life and life processes in general. The roles of enzymes are identified by identifying what type of enzymes they are and what processes or reactions they aid in . For the preliminary studies, various computational tools can be used . We performed computational analysis using several different programs as shown in Figure 1 . The Protein Data bank (PDB1) first tells us about the quantitative properties and the structure of the enzyme . Each structure in the PDB is assigned a four character alphanumeric identifier, called the PDB ID, and the focus of our current study is the structure with PDB ID 4 Q7 Q (Figure 2 ). The active site of an enzyme is the region where the substrate molecules bind and undergo a chemical reaction . This active site consists of various amino acid residues that interact with the substrate forming temporary bonds and residues which catalyze the reaction of the substrate . The Enzyme Commission (EC) number is a numerical classification scheme for the enzyme, and this can tell us a lot about the given enzyme . The program Moltimate2can be used to compare 3 D protein structures with a library of enzyme active sites, to predict whether the query structure (often a protein of unknown function) aligns with an enzyme of known function . These active sites can be better visualized with visualization software such as PyMOL3. The protein sequence can be run through BLAST4to find any other protein of known function which may help predict the potential function of the unknown protein . Similarly, more information can be found with the help of other tools such as PFam5and Dali6. PFam is a database of protein families, where families are sets of protein regions that share a significant degree of sequence similarity, thereby suggesting homology . Dali is used to align the backbone for entire protein structures . Finally , SwissDock7a protein ligand docking program can be used to predict the position and orientation of a ligand or substrate of our choice when it is bound to a protein receptor or enzyme . Our computational analysis led us to conclude that the enzyme 4 Q7 Q was a possible lipase with specificity to ester substrates . Kinetic testing was done to test this hypothesis . Enzyme kinetics is the study of the rates of enzyme catalyzed chemical reactions . A measured rate can be used to derive information about the amount of enzyme present in a reaction sample, or about the effects of different experimental conditions on rate . The Michaelis Menten model for enzyme activity treats an enzyme catalyzed reaction as a simple two step process ; the first, association of substrate and enzyme to form enzyme substrate complex, and the second, conversion of substrate to product . Plotting the Michelis Menten graph will help us verify whether the enzyme has an affinity for a certain substrate . TESTING FOR ESTERASE ACTIVITY IN ENZYME 4Q7Q Namrata Pokharel and Julia R. Koeppe Department of Chemistry, SUNY Oswego, NY MethodsThe enzyme was analyzed with the help of structure viewing and alignment programs, and the alignments compared to various known enzymes in order to classify it and predict the potential functions . Computational analysis revealed it to be a potential lipase with specific affinity towards ester substrates . A plasmid containing the gene for 4 Q7 Q was successfully transformed in E . Coli by creating a surface that only bacteria with ampicillin resistance can survive and reproduce upon. Overnight Expression TB was then used for protein expression . Bacteria grew overnight by the accelerated process of TB . The cells were then spun down and the His tagged protein was purified by metal affinity chromatography after cell lysis . SDS PAGE was used to verify the presence of protein . Buffer exchange was performed to reduce the salt content and remove the imidazole and obtain as high a concentration of protein as possible . The Bradford assay was then carried out to quantify the amount of protein in samples . Enzyme kinetics experiments were performed using a chromogenic substrate . Trial and error was used to determine conditions for carrying out assays to verify whether 4 Q7 Q follows Michaelis Menten kinetics for any substrate .ResultsComputational Analysis: All of the alignments performed indicate that 4 Q7 Q is a hydrolase with specificity for ester bonds . The 4 Q7 Q amino acid sequence and structure align with some acetyl esterases and some acyl esterases . The best active site alignment was to the 1 BWR structure. 1 BWR is described as an acetylhydrolase on the PDB (Figure 3 ). SwissDock was used to identify a possible binding site for the ligand propanoic acid (PPI) in 4 Q 7 Q (Figure 4 ) . This ligand is bound to various esterase structures on the PDB . Purification of 4 Q 7 Q : 4 Q7 Q was successfully purified using NiNTA agarose as verified by SDS PAGE analysis (Figure 5 ). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 2 4 6 8 10 12 14 16 18 20AbsorbanceTime (minutes)Absorbance Vs Time for p nitrophenyl butyrate and 4Q7Q Figure 6: Absorbance Vs Time plot for p nitrophenyl butyrate hydrolysis . The increase in absorbance as a function of time indicates that the yellow colored product is being produced upon hydrolysis of the substrate . Figure 7: Michaelis Menten plots (V0vs [S]) for reaction of enzyme 4Q7Q with p nitrophenyl butyrate (left) and with p nitrophenyl acetate (right). Figure 4. The active site of 4Q7Q is shown in blue and PPI from 5AOA is shown in purple . This ligand has a binding affinity of 4. 1, a RMSD/ ub of 2. 437, and a RMSD/ lb of 2. 027. The PPI from 5LY 1 is shown in green and this ligand has a binding affinity of 4 . 1, a RMSD/ ub of 2. 49, and a RMSD/ lb of 2. 114. Figure 3. Active Site Alignment with 4Q7Q in green and 1BWR in red . The RMSD values for 5 residues with 35 atoms is 0. 496, 5 residues with 0. 459, and 5 residues with 9 atoms is 0. 509 . The Levenshtein distance is 05 and the EC class of 1BWR is 3. 1. 1. 47. Figure 1. Process used for characterization of proteins of ‘‘unknown function’’ . The top two boxes focus on in silico methods, followed by in biblio and in vitro steps [1, 2]. Figure 2. The structure of 4Q7Q from the PDB . Figure 5. SDS PAGE analysis of the purification of 4Q7Q by metal affinity chromatography . The His tagged 4Q7Q was separated from other proteins by washing the column with buffers containing increasing amounts of imidazole . Elution fraction 2 and 3 were combined and kept for kinetic testing .



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Introduction: Methods: Results: Background The historic use of the insecticide lead arsenate throughout the US has left the biologically harmful metal, lead (Pb), to persist in soils today. The upper/surface horizons, or layers, of a soil profile contain more of the organic and sandy soils. These horizons also tend to contain higher lead (Pb) concentrations 1 . Children are at the greatest risk of Pb toxicity, putting them in their mouth 2 . This early childhood Pb exposure has been linked to neuro cognitive deficits and social problems in adulthood 2 . Study Area Rice Creek Field Station was established in 1965 66 by SUNY Oswego 3 . Prior to this, much of the surrounding area was pasture and farmland, including an abandoned orchard 3 . The shared area of Figure 1 locates this abandoned orchard area 3 . Figure 1: Modified Rice Creek Field Station Map 3 . Shaded orange area represents the abandoned orchard location. Sample Preparation and Digestion Soil samples were collected using a Forestry Suppliers Tubular Soil Sampler. Segments were separated into 2 cm increments with respect to depth below the soil surface. Segments were collected in labeled plastic sandwich bags. Once back in the lab, segments were left in paper bags to air dry over multiple days. Now dried soil segments were ground using a mortar and pestle, with visible rock fragments that were unable to be broken removed 4,5 . 2 gram portions of each segment were then divided into fused silica quartz crucibles 4,5 . These portions were heated at 475 overnight to ash the organic components of the soil 4,5 . Ashed segment samples were digested in 10 mL of 7.5 M HNO 3 solution 4,5 . Each digestion was performed for 1 hour directly in the fused silica quartz crucibles, on 4,5 . Digested samples were gravity filtered using Fischerbrand P5 filter paper in 50 mL volumetric flasks and diluted to the mark using deionized water 5 . These sample solutions were then transferred to low density polyethylene (LDPE) bottles for storage. Standard Solution Preparation Standard solutions were prepared using CertiPur 1000 ppm Lead AA standard, diluted to form a 200 ppm Pb concentrated standard solution. A blank solution of 0.075 M HNO 3 was made at a large volume. 10 ppm, 25 ppm, 50 ppm, and 100 ppm Pb standard solutions were made by portioning the 200 ppm Pb standard solution. Each Pb standard solution was diluted using the 0.075 M HNO 3 blank solution. Conclusion and Discussion An Agilent Technologies 200 Series AA instrument was used to determine absorbance values of all Pb solutions. Figure 3 illustrates the calibration curve of the Pb standards, which was used to determine the Pb concentration of each sample solution for Locations A and B. Figure 4 illustrates the determined Pb concentrations of each 2 g segment before dilution with deionized water for Locations A and B. More exemplified by Location B, Pb concentrations can be seen to decrease at deeper depths of soil. This is likely due to a transition between soil horizons, to deeper horizons containing lower Pb concentrations. If cores of a deeper depth could be taken, this decrease in Pb concentration and deeper horizons would likely be more profound. Due to lower absorbance readings of sample solutions, determined Pb concentrations in the original soil segment samples are rounded. Determination of Pb in orchard soil samples at the Rice Creek Field Station Warren Kincaid, Dr. Jeffery Schneider Acknowledgements: Thank you Dr. Thomas Brown, Dr. Vadoud Niri , and Kristin Gublo for your help throughout these past fall and spring semesters. References: 1 Durkee , J.; Bartrem , C.; Moller, G.; Legacy lead arsenate soil contamination at childcare centers in Yakima Valley, Central Washington, USA. Chemosphere. 2017 , 168, 1126 1135 2 ToxGuide for Lead. U.S. Department of Health and Human Services Agency for Toxic Substances and Disease Registry. 2020 , https://wwwn.cdc.gov/TSP/ToxProfiles/ToxProfiles.aspx?id=96&tid=22 3 About | Rice Creek Field Station https://www.oswego.edu/rice creek/about (accessed Apr 6, 2021). 4 , A.; , M.; Comparison of Two Extractions for Determination of Trace Metals in Soil by Atomic Absorption Spectrometry. Arh . Hig . Rada Toksikol . 1998 , 49, 327 334 5 Schneider, J. A. The Spectrophotometric Determination of Lead with 5/10,15,20 Tetra(4 N 8ulfoethyIpyridinium) porphine Using Merging Zones Flow Injection Analysis. Ph.D. Thesis, Dartmouth College, June 1992 6 Ander, E. L.; Johnson, C. C; Cave, M. R.; Palumbo Roe, B.; Nathanail , C. P.; Lark, R. M.; Methodology for the determination of normal background concentrations of contaminants in English soil. Sci. Total Environ. 2013 , 454 455 , 604 618 7 Anderson, P.; Soil and Soil Dynamics Bozeman Science. https://www.youtube.com/watch?v=mg7XSjcnZQM&feature=emb_logo , (accessed Sept. 25, 2020), educational Youtube video 8 Rashid, M. H.; Fardous , Z.; Chowdhury, M.A.Z.; Alam , M. K.; Bari, M. L.; Moniruzzaman , M.; Gan, S.H.; Determination of heavy metals in the soils of tea plantations and in fresh and processed tea leaves: an evaluation of six digestion methods. Chem Cent J. 2016 , 10 , 7 9 Pociecha , M.; Lestan , D.; Recycling of EDTA solution after soil washing of Pb, Zn, Cd and As contaminated soil. Chemosphere. 2012 , 86, 843 846 Figure 3: Calibration curve of Pb standard solutions. Figure 4: Pb concentrations in soil segments with respect to depth below soil surface.