DOCSLIB.ORG

Role of Proinsulin Self-Association in Mutant INS Gene-Induced Diabetes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Role of Proinsulin Self-Association in Mutant INS Gene-Induced Diabetes Page 1 of 43 Diabetes Role of Proinsulin Self-Association in Mutant INS gene-induced Diabetes of Youth Jinhong Sun1*, Yi Xiong1*, Xin Li2, Leena Haataja1, Wei Chen1,3, Saiful A. Mir4, Li Lv2, Rachel Madley1, Dennis Larkin1, Arfah Anjum1, Balamurugan Dhayalan5, Nischay Rege5, Nalinda D. Wickramasinghe5, Michael A. Weiss5, Pamela Itkin-Ansari4,8, Randal J. Kaufman6, David A. Ostrov7, Peter Arvan1¶, Ming Liu1,2¶ 1. Division of Metabolism, Endocrinology & Diabetes, the University of Michigan Medical School, Ann Arbor, MI 48105 USA 2. Department of Endocrinology and Metabolism, Tianjin Medical University General Hospital, Tianjin 300052, China 3. Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China 4. Development, Aging and Regeneration Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037 USA 5. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis IN 46202 USA 6. Degenerative Diseases Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA 92037 USA 7. Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610 USA 8. Department of Pediatrics, University of California, San Diego, La Jolla, CA 92093 USA * Authors contributed equally to this work. ¶ To whom correspondence should be addressed: Ming Liu, Department of Endocrinology and Metabolism, Tianjin Medical University General Hospital, 154 Ann Anshan road, Heping district, Tianjin 300052, China. E-mail: [email protected]; Peter Arvan, Division of Metabolism, Endocrinology & Diabetes, the University of Michigan, Brehm Tower, 1000 Wall Street, Ann Arbor, MI 48105 E-mail: [email protected]; 1 Diabetes Publish Ahead of Print, published online March 5, 2020 Diabetes Page 2 of 43 Abstract Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin in the endoplasmic reticulum (ER) drive the molecular pathogenesis of Mutant-INS-gene induced Diabetes of Youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, proinsulin-WT dimerizes in the ER. Here, we suggest that the normal proinsulin-proinsulin contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that proinsulin Tyr-B16, which is a key residue in normal proinsulin dimerization, helps confer dominant-negative behavior of MIDY mutant proinsulin-C(A7)Y. Substitutions of Tyr-B16 with ether Ala, Asp, or Pro in proinsulin-C(A7)Y each decrease the abnormal interactions between the MIDY mutant and proinsulin-WT, rescuing proinsulin-WT export, limiting ER stress, and increasing insulin production in -cells and human islets. This study reveals the first evidence indicating that noncovalent proinsulin-proinsulin contact initiates dominant-negative behavior of misfolded proinsulin, pointing to a novel therapeutic target to enhance proinsulin-WT export and increase insulin production. 2 Page 3 of 43 Diabetes Introduction Proinsulin misfolding, endoplasmic reticulum (ER) stress, and decreased -cell mass are key features of -cell failure and diabetes in the autosomal-dominant diabetes known as Mutant-INS-gene induced Diabetes of Youth (MIDY) (1-3). All MIDY patients carry a mutation in only one of two INS alleles (3-6), and, in principle, one normal INS allele should be sufficient to maintain normoglycemia (7). The underlying mechanism of MIDY is thought to involve abnormal interactions of misfolded mutant proinsulin with bystander proinsulin-WT in the ER, impairing the ER export of proinsulin-WT and decreasing production of bioactive mature insulin (8,9). However, to date, how these interactions are initiated remains largely unknown. Interestingly, the clinical spectrum of MIDY ranges from neonatal-onset severe insulin deficient diabetes to relatively late- onset mild diabetes (1,2), suggesting that different proinsulin mutants may behave differently in their folding processes, as well as their ability to interact and obstruct bystander proinsulin-WT in the ER. In this study, we report that a proline (Pro) substitution for tyrosine residue at the 16th position of the proinsulin B chain (Tyr-B16) [i.e., proinsulin-Y(B16)P] results in dramatic proinsulin misfolding in the ER, and yet is unable to interact with bystander proinsulin-WT in the ER, and is unable to impair the ER export of proinsulin-WT or to block its ability to form mature insulin. Interestingly, Y(B16)P disrupts the main α-helix in the proinsulin B-domain. Normally, proinsulin uses this B-domain α-helix in order to homodimerize in the ER (10). Thus, we hypothesize that cross-dimerization between 3 Diabetes Page 4 of 43 MIDY mutant and proinsulin-WT may be required to initiate their abnormal interaction that impairs the intracellular transport of proinsulin-WT, resulting in insulin deficiency that leads to diabetes (11). Structural evidence strongly implicates Tyr-B16 as a key residue for normal proinsulin dimerization (12). With this in mind, we have mutagenized this residue to either aspartic acid [Y(B16D)] or alanine [Y(B16)A]; both substitutions were entirely innocuous when introduced into proinsulin-WT. Notably, however, when these substitutions were introduced into the MIDY-causing Akita proinsulin-C(A7)Y, each could function as an intra-allelic suppressor, limiting abnormal interactions with proinsulin-WT; alleviating dominant-negative behavior on proinsulin-WT intracellular trafficking; and increasing insulin production in pancreatic -cell lines and human islets. This study provides insight that protecting the proinsulin-proinsulin contact surface might be a potential druggable target to limit abnormal proinsulin interactions and prevent/delay the development and progression of MIDY. 4 Page 5 of 43 Diabetes Materials and Methods Materials Guinea pig polyclonal anti-porcine insulin, human insulin-specific RIA kit, human proinsulin-specific RIA kit, and mouse insulin ELISA kit were from Millipore (Billerica MA, USA); Guinea pig polyclonal anti-human insulin was from Novus (Centennial, CO, USA); Rabbit polyclonal anti-mouse proinsulin C-peptide (EVEDPQVAQLELGGGPGAGDLQTLALEVAQQ) was raised by Bioworld (Nanjing, China); Mouse monoclonal anti-human proinsulin C-peptide—A-chain junction (GSLQKRGIVE) was raised by Abmart (Shanghai, China) as previously described (13) ; Rabbit anti-Myc and anti-GFP were from Immunology Consultants Laboratories (Portland, OR, USA); Rabbit anti-BiP was a gift from Dr. P.S. Kim (U. Cincinnati); 35S-amino acid mixture was from PerkinElmer (Waltham, MA, USA); dithiothreitol (DTT), protein A- agarose, digitonin, N-ethylmaleimide (NEM), brefeldin A, and all other chemical reagents were from Sigma-Aldrich (St. Louis, MO, USA); 4-12% gradient SDS-PAGE employed NuPage gels, Met/Cys-deficient Dulbecco’s modified Eagle’s medium (DMEM) and all other tissue culture reagents were from Invitrogen (Thermo Fisher, Waltham, MA, USA); tris-tricine-urea-SDS-PAGE was performed as described previously (14). Synthesis and purification of single-chain insulin analogs, and NMR Single-chain TyrB16-DesDi and AspB16-DesDi insulin analogs were synthesized using a Tribute Peptide Synthesizer with preprogrammed solid-phase Fmoc protocol (15). The synthesis was carried out on a 0.1-mmol scale using H-Asn(Trt)-HMPB-ChemMatrix resin 5 Diabetes Page 6 of 43 (0.46 mmol/g loading). Side chain protection was: Asn(Trt), Arg(Pbf), Asp(OtBu) Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Tyr(tBu). DIC/ 6- chloro-HOBt in 1:2 Dichloromethane/DMF was used for the coupling and 20% piperidine in DMF for Nα-Fmoc-deprotection. The product peptide was then cleaved from the resin and simultaneously deprotected by subjecting it to TFA/TIPS/water/DODT/anisole (90: 2.5: 2.5: 2.5: 2.5 v/v) for 2.5 h at ambient temperature; the crude product was precipitated with ice-cold ether, washed, and dried. For folding, 100 mg AspB16-DesDi crude polypeptide (corresponding to 18 µmol), cysteine hydrochloride (43mg, 2 mM) and glycine (270 mg, 20 mM) were placed in 600-mL glass beaker and distilled, deionized water added to a 0.1 mM final concentration. The pH of the solution was adjusted to 10.5, and reaction was stirred open to air at 4° C for 16 h. Desired folded product was purified both by preparative HPLC, and by analytical reverse-phase HPLC on a C8 Proto (4.6x250 mm) 300 Å, 5 μm, Higgins Analytical Inc. column, using 25-50% Solvent B (0.1% TFA in acetonitrile) in Solvent A (0.1% TFA in water) over 35 min at a flow rate of 1.0 ml/min (Waters 1525). Protein elution was monitored at 215 and 280 nm (Waters 2489 absorbance detector) and masses were confirmed by MALDI-TOF. Normal insulin helical structure was confirmed by far-UV CD spectra from 190-250 nm (16), and normal thermodynamic stability was established by guanidine hydrochloride-induced denaturation (monitored by CD at 222 nm). 1H-NMR spectra were acquired on protein solutions (0.5 mg in 300 L D2O; direct meter reading pD 7.6) at 25° C using a Bruker AVANCE 700 MHz spectrometer. Synthesis and purification of two-chain insulin analogs, and size exclusion chromatography. 6 Page 7 of 43 Diabetes The non-standard amino acid Ornithine (Orn) was employed at position B29 in place of lysine to preserve the side-chain amino group without susceptibility to tryptic cleavage (16). Two-chain insulin analogs ([TyrB16, OrnB29]-insulin and [AspB16, OrnB29]-insulin) were prepared by trypsin-catalyzed cleavage of human insulin or AspB16-DesDi single-
Load more

Recommended publications
  • Buffers a Guide for the Preparation and Use of Buffers in Biological Systems Calbiochem® Buffers a Guide for the Preparation and Use of Buffers in Biological Systems
    Buffers A guide for the preparation and use of buffers in biological systems Calbiochem® Buffers A guide for the preparation and use of buffers in biological systems Chandra Mohan, Ph.D. EMD, San Diego, California © EMD, an affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. A word to our valued customers We are pleased to present to you the newest edition of Buffers: A Guide for the Preparation and Use of Buffers in Biological Systems. This practical resource has been especially revamped for use by researchers in the biological sciences. This publication is a part of our continuing commitment to provide useful product information and exceptional service to you, our customers. You will find this booklet a highly useful resource, whether you are just beginning your research work or training the newest researchers in your laboratory. Over the past several years, EMD Biosciences has clearly emerged as a world leader in providing highly innovative products for your research needs in Signal Transduction, including the areas of Cancer Biology, Alzheimer’s Disease, Diabetes, Hypertension, Inflammation, and Apoptosis. Please call us today for a free copy of our LATEST Catalog that includes tools for signal transduction and life science research. If you have used our products in the past, we thank you for your support and confidence in our products, and if you are just beginning your research career, please call us and give us the opportunity to demonstrate our exceptional customer and technical service. Corrine Fetherston Sr. Director, Marketing ii Table of Contents: Why does Calbiochem® Biochemicals Publish a Booklet on Buffers? .
    [Show full text]
  • Biological Buffers and Ultra Pure Reagents
    Biological Buffers and Ultra Pure Reagents Are MP Buffers in your corner? One Call. One Source. A World of Ultra Pure Biochemicals. www.mpbio.com Theoretical Considerations Since buffers are essential for controlling the pH in many Since, under equilibrium conditions, the rates of dissociation and biological and biochemical reactions, it is important to have a association must be equal, they may be expressed as: basic understanding of how buffers control the hydrogen ion concentration. Although a lengthy, detailed discussion is impractical, + - k1 (HAc) = k2 (H ) (Ac ) some explanation of the buffering phenomena is important. Or Let us begin with a discussion of the equilibrium constant (K) for + - weak acids and bases. Acids and bases which do not completely k1 = (H ) (Ac ) dissociate in solution, but instead exist as an equilibrium mixture of k (HAc) undissociated and dissociated species, are termed weak acids and 2 bases. The most common example of a weak acid is acetic acid. If we now let k1/k2 = Ka , the equilibrium constant, the equilibrium In solution, acetic acid exists as an equilibrium mixture of acetate expression becomes: ions, hydrogen ions, and undissociated acetic acid. The equilibrium between these species may be expressed as follows: + - Ka = (H ) (Ac ) k1 (HAc) + - HAc ⇌ H + Ac which may be rearranged to express the hydrogen ion concentration k2 in terms of the equilibrium constant and the concentrations of undissociated acetic acid and acetate ions as follows: where k1 is the dissociation rate constant of acetic acid to acetate and hydrogen ions and k is the association rate constant of the ion 2 (H+) = K (HAc) species to form acetic acid.
    [Show full text]
  • Electrophoresis Tech Note 5956
    electrophoresis tech note 5956 Precision Plus Protein™ Dual Xtra Standards — New Protein Standards with an Extended Range from 2 to 250 kD Sansan Lin, Jonathan E. Kohn, Joseph Siino, Dennis C. Yee, and the standard were used to estimate the molecular weights Michael Urban, Bio-Rad Laboratories, Inc., 6000 James Watson Drive, of five known sample proteins. The results presented here Hercules, CA 94547 USA. demonstrate that the Precision Plus Protein Dual Xtra Introduction standards offer a convenient and reliable method to estimate The Precision Plus Protein Dual Xtra standards are expanded the molecular weight of proteins on a variety of gel types. range molecular weight standards for protein electrophoresis composed of 12 recombinant proteins and peptides with Methods molecular weights ranging from 2 to 250 kD. The Dual Xtra Sample Preparation Precision Plus Protein Dual Xtra standards were used standards are prestained with two colors for easy band according to manufacturer’s specifications. A total of referencing during electrophoresis and blot transfer. 10 μl of Dual Xtra standards was loaded onto each lane. Protein standards are tools commonly used with one- All other purified proteins were purchased from Sigma- dimensional electrophoresis to determine the molecular Aldrich. Sample proteins were suspended in Laemmli sample weight of unknown proteins (Bio-Rad Bulletin 3133). In buffer containing b-mercaptoethanol (Bio-Rad Laboratories, polyacrylamide gel electrophoresis (PAGE), proteins are Inc.) and heated at 85°C for 6 min prior to loading. driven through the gel under an electric field, during which Gel Electrophoresis and Molecular Weight Analysis the gel matrix acts as a sieve to separate proteins based on For migration analysis a total of 10 μl of Precision Plus size (SDS-PAGE), or charge and shape (native PAGE).
    [Show full text]
  • Protein Gel Electrophoresis Technical Handbook Separate Transfer Detect 2
    Western blotting Protein gel electrophoresis technical handbook separate transfer detect 2 Comprehensive solutions designed to drive your success Protein gel electrophoresis is a simple way to separate proteins prior to downstream detection or analysis, and is a critical step in most workflows that isolate, identify, and characterize proteins. We offer a complete array of products to support rapid, reliable protein electrophoresis for a variety of applications, whether it is the first or last step in your workflow. Our portfolio of high-quality protein electrophoresis products unites gels, stains, molecular weight markers, running buffers, and blotting products for your experiments. For a complete listing of all available products and more, visit thermofisher.com/separate For orderingordering informationinformation referrefer to pagepage XX.XX ForForqr quickquickrk referencereferencee on the protocolprotocol pleasepleasere referrefertr totopo pagepage XX. Prepare samples Choose the electrophoresis Select precast gel and select buffers Select the standard chamber system and power supply Run the gel Stain the gel Post stain 3 Contents Electrophoresis overview 4 Select precast gel Gel selection guide 8 Gels 10 Prepare samples and select buffers Sample prep kits 26 Buffers and reagents 28 Buffers and reagents selection guide 29 Select the standard Protein ladders 34 Protein standards selection guide 36 Choose the electrophoresis chamber system and power supply Electrophoresis chamber systems 50 Electrophoresis chamber system selection guide 51
    [Show full text]
  • Tricine–SDS-PAGE Hermann Schägger
    PROTOCOL Tricine–SDS-PAGE Hermann Schägger Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, Haus 26, D-60590 Frankfurt, Germany. Correspondence should be addressed to H.S. ([email protected]). Published online 12 May; corrected online 10 August 2006; doi:10.1038/nprot.2006.4 Tricine–SDS-PAGE is commonly used to separate proteins in the mass range 1–100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa. The concentrations of acrylamide used in the gels are lower than in other electrophoretic systems. These lower concentrations facilitate electroblotting, which is particularly crucial for hydrophobic proteins. Tricine–SDS-PAGE is also used preferentially for doubled SDS-PAGE (dSDS-PAGE), a proteomic tool used to isolate extremely hydrophobic proteins for mass spectrometric identification, and it offers advantages for resolution of the second otocols dimension after blue-native PAGE (BN-PAGE) and clear-native PAGE (CN-PAGE). Here I describe a protocol for Tricine–SDS-PAGE, r ep which includes efficient methods for Coomassie blue or silver staining and electroblotting, thereby increasing the versatility of r the approach. This protocol can be completed in 1–2 d. INTRODUCTION 1 e.com/natu r Glycine–SDS-PAGE (also known as Laemmli–SDS-PAGE) and because the stacking limit in the Laemmli system is too high, Tricine–SDS-PAGE2,3, based on glycine-Tris and Tricine-Tris and small proteins usually appear as smearing bands near the .natu buffer systems, respectively, are the commonly used SDS electro- gel front. In a less convenient way, however, the small protein w phoretic techniques for separating proteins.
    [Show full text]
  • Western Blotting
    ptglab.com 1 The Complete Guide To WESTERN BLOTTING www.ptglab.com 2 Western Blotting ptglab.com 3 WELCOME About this guide This booklet aims to give you a complete guide on how to perform a Western blot that produces optimal results. The booklet includes original Western blot lab protocols, useful technical tips, and troubleshooting. This guide will take you through the experiment step by step, enabling you to get the most from your research. What’s Inside 6–9 Standard Western Blot Protocol 10–11 How To Optimize Your Western Blot 12–13 SDS-PAGE Gel Recipes 14–15 How To Optimize Your Results With Low MW Proteins 16 Tricine Gel Recipe For Low MW Proteins 17 Choosing The Right Lysis Buffer 18–23 Troubleshooting 26–30 Loading Control Antibodies 31 Contact Us 4 Western Blotting THE BENCHMARK IN ANTIBODIES Since the day it was founded, Proteintech®* has been making all of its products to the highest standards possible whilst taking complete responsibility for the quality of each product. • Proteintech makes every single antibody in its 12,000+ catalog. • Each Proteintech product is unique and cannot be bought under a different label. • Antibodies are detected with siRNA-treated samples to demonstrate specificity. • It works in every single species and application or get a full money-back refund. Proteintech has over 12,000 antibodies in its extensive catalog, all fully validated and available for next day delivery. www.ptglab.com *Proteintech® and the Proteintech logo are trademarks of Proteintech Group registered in the US Patent and Trademark Office. ptglab.com 5 6 Western Blotting Standard Western Blot PROTOCOL SDS-PAGE 1.
    [Show full text]
  • GENECTIVE SA Maize Event VCO-Ø1981-5 Page 2 of 163
    GENECTIVE SA Maize event VCO-Ø1981-5 Page 2 of 163 COMPANY NAMES GENECTIVE S.A., an affiliate of KWS and VCO Participation, an affiliate of Vilmorin & Cie, is a company organized and existing under the laws of France, having its head office at 1 rue Limagrain, 63720 Chappes, France, SIREN Number 513 533 612, RCS Clermont-Ferrand. On July 1, 2008, Athenix Corp. and Vilmorin & Cie entered into an agreement for the development of maize tolerant to glyphosate and resistant to insects. Under this agreement, Athenix Corp. has licensed to Vilmorin & Cie the right to use worldwide the genes they discover and own for the above traits. On June 23, 2011 Vilmorin & Cie has transferred the above agreement to its affiliate GENECTIVE S.A. On November 2, 2009 Athenix Corp., a biotechnology company that develops novel products and technology for agricultural applications, became an affiliate of Bayer CropScience LP. GENECTIVE S.A. and Bayer CropScience are continuing the collaboration initiated in 2008 between Vilmorin & Cie and Athenix Corp. Some of the activities described in this petition were undertaken before the acquisition of Athenix Corp. by Bayer CropScience LP and based on the agreement between Athenix Corp. and Vilmorin & Cie. CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION GENECTIVE SA Maize event VCO-Ø1981-5 Page 3 of 163 RELEASE OF INFORMATION The information in this petition is being submitted by GENECTIVE S.A. for review by the USDA as part of the regulatory process. By submitting this information, GENECTIVE S.A. does not authorize its release to any third party except to the extent it is requested under the Freedom of Information Act (FOIA), 5 U.S.C., Section 552 and 7 CFR 1, covering all or some of this information.
    [Show full text]
  • A Versatile Electrophoresis System for the Analysis of High and Low Molecular Weight Proteins
    A versatile electrophoresis system for the analysis of high and low molecular weight proteins Christophe Tastet1, Pierre Lescuyer1, Hélène Diemer2, Sylvie Luche1, Alain van Dorsselaer2 and Thierry Rabilloud1 1: CEA- Laboratoire de Bioénergétique Cellulaire et Pathologique, EA 2943, DRDC/BECP CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9, France 2: Laboratoire de Spectrométrie de Masse Bio-Organique, UMR CNRS 7509, ECPM, 2( rue Becquerel, 67008 Strasbourg Cedex Correspondence : Thierry Rabilloud, DRDC/BECP CEA-Grenoble, 17 rue des martyrs, F-38054 GRENOBLE CEDEX 9 Tel (33)-4-38-78-32-12 Fax (33)-4-38-78-51-87 e-mail: Thierry.Rabilloud@ cea.fr Abbreviations: AMPSO: (1,1 dimethyl-2-hydroxy ethyl)-3-aminp-2-hydroxy-propane sulfonic acid Abstract A new, versatile, multiphasic buffer system for high resolution sodium dodecyl sulfate- polyacrylamide gel electrophoresis of proteins in the relative molecular weight Mw range of 300,000-3000 Da is described. The system, based on the theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins in the above Mw range. The buffer system uses taurine and chloride as trailing and leading ion, respectively, and Tris, at a pH close to its pKa, as the buffering counter ion. Coupled with limited variation in the acrylamide concentration, this electrophoresis system allows to tailor the resolution in the 6-200 kDa Mw range, with minimal difficulties in the post electrophoretic identification processes. 1 Introduction In almost all 2D electrophoresis systems, the SDS PAGE dimension uses the classical Tris- chloride-glycine discontinuous system introduced by Ornstein and Davis [1].
    [Show full text]
  • Acid Dissociation Constants and Related Thermodynamic Functions of Protonated 2,2-Bis(Hydroxymethyl)-2,2’,2”- Nitrilotriethanol (BIS-TRIS) from (278.15 to 328.15) K
    Journal of Biophysical Chemistry, 2014, 5, 118-124 Published Online August 2014 in SciRes. http://www.scirp.org/journal/jbpc http://dx.doi.org/10.4236/jbpc.2014.53013 Acid Dissociation Constants and Related Thermodynamic Functions of Protonated 2,2-Bis(Hydroxymethyl)-2,2’,2”- Nitrilotriethanol (BIS-TRIS) from (278.15 to 328.15) K Rabindra N. Roy, Lakshmi N. Roy, Katherine E. Hundley, John J. Dinga, Mathew R. Medcalf, Lucas S. Tebbe, Ryan R. Parmar, Jaime A. Veliz Hoffman Department of Chemistry, Drury University, Springfield, USA Email: [email protected] Received 14 May 2014; revised 12 June 2014; accepted 11 July 2014 Copyright © 2014 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ Abstract Thermodynamic dissociation constants pKa of 2,2-bis(hydroxymethyl)-2,2’,2”-nitrilotriethanol have been determined at 12 temperatures from (278.15 to 328.15) K including the body temper- ature 310.15 K by the electromotive-force measurements (emf) of hydrogen-silver chloride cells without liquid junction of the type: Pt(s), H2(g), 101.325 kPa|BIS-TRIS (m) + BIS-TRIS·HCl (m)| AgCl(s), Ag(s), where m denotes molality. The pKa values for the dissociation process of BIS- + + TRIS·H + H2O = H3O + BIS-TRIS given as a function of T in Kelvin (K) by the equation pKa = 921.66 (K/T) + 14.0007 − 1.86197 ln(T/K). At 298.15 and 310.15 K, the values of pKa for BIS-TRIS were found to be 6.4828 ± 0.0005 and 6.2906 ± 0.0006 respectively.
    [Show full text]
  • HR2-081 PEG/Ph 2 Documents
    PEG/pHTM 2 Solutions for Crystal Growth User Guide HR2-081 (pg 1) Applications tration is 5 to 25 mg/ml in dilute (25 mM or less) buffer. For initial screens, Crystallization screen for biological macromolecules, where Polyethylene glycol the sample should be free of unnecessary additives in order to observe the effect 4,000 is the primary reagent, and multiple buffer agents the secondary, sam- of the PEG/pH 2 reagents. However, agents that promote and preserve sample pling a neutral to alkaline pH, or, sampling a broad, acidic to alkaline range of solubility, stability, and homogeneity can and should be included in the sample pH when used together with PEG/pH. buffer. For additional sample preparation recommendations see Hampton Re- search Crystal Growth 101 - Preliminary Sample Preparation. Features • Samples pH 6.4 - 9.6 in 0.2 M buffer Manual Method - Sitting Drop Vapor Diffusion • Samples pH 3.5 - 9.6 in 0.2 M buffer when used with PEG/pH 1. Using four 24 well sitting drop crystallization plates, pipet the recommended • Polyethylene glycol 4,000 - primary reagent volume (typically 500 to 1,000 microliters) of crystallization reagent from the • 10 unique buffers, 0.2 M - secondary reagent tubes into the crystallization plates. PEG/pH 2 reagents 1 through 24 into the • 20 unique buffers, 0.2 M - secondary reagent when used with PEG/pH first plate and reagents 25 through 48 into the second plate. Use clean pipette • Vapor diffusion, microbatch, free interface diffusion tips, a new tip for each of the 48 reagents. • Useful for screening samples where a neutral to alkaline pH range is desired 2.
    [Show full text]
  • Biological Buffers and Ultra Pure Reagents
    Biological Buffers and Ultra Pure Reagents One Call. One Source. A World of Ultra Pure Biochemicals. Are MP Buffers in your corner? Find it at fishersci.com Theoretical Considerations Since buffers are essential for controlling the pH in many Since, under equilibrium conditions, the rates of dissociation and biological and biochemical reactions, it is important to have a association must be equal, they may be expressed as: basic understanding of how buffers control the hydrogen ion concentration. Although a lengthy, detailed discussion is impractical, + - k1 (HAc) = k2 (H ) (Ac ) some explanation of the buffering phenomena is important. Or Let us begin with a discussion of the equilibrium constant (K) for + - weak acids and bases. Acids and bases which do not completely k1 = (H ) (Ac ) dissociate in solution, but instead exist as an equilibrium mixture of k (HAc) undissociated and dissociated species, are termed weak acids and 2 bases. The most common example of a weak acid is acetic acid. If we now let k1/k2 = Ka , the equilibrium constant, the equilibrium In solution, acetic acid exists as an equilibrium mixture of acetate expression becomes: ions, hydrogen ions, and undissociated acetic acid. The equilibrium between these species may be expressed as follows: + - Ka = (H ) (Ac ) k1 (HAc) HAc H+ + Ac- ⇌ which may be rearranged to express the hydrogen ion concentration k2 in terms of the equilibrium constant and the concentrations of undissociated acetic acid and acetate ions as follows: where k1 is the dissociation rate constant of acetic acid to acetate and hydrogen ions and k is the association rate constant of the ion 2 (H+) = K (HAc) species to form acetic acid.
    [Show full text]
  • User Guide for Goldbio Buffers
    User Guide for GoldBio Buffers GoldBio © 2015 All Rights Reserved This Publication is a creation of Gold Biotechnology and is intended as a sourcebook for research laboratories. This publication may not be redistributed or copied for commercial use. Procedure for use with Gold Biotechnology Buffers. Table of Contents Introduction: .................................................................................................................................................3 GoldBio Buffers Grouped By Structural Family:............................................................................................4 Acetamido Family (ACES, ADA).................................................................................................................4 Bis(2-hydroxyethyl)amine Family (Bis-Tris, BES, Bicine)...........................................................................6 Cyclohexylamino Family (CHES, CAPS)......................................................................................................9 Morpholinic Family (MES, MOPS, MOPSO) ............................................................................................11 Piperazinic Family (PIPES, HEPES, HEPPSO) ............................................................................................14 Tris Family (Tris, Bis-Tris, TES, TAPS, Tricine) ..........................................................................................18 Other buffers(Imidazole, Glyclglycine, Boric Acid, PBS tablets) .............................................................22
    [Show full text]