University:
Boston University
Course:
CAS BI 527 | Biochemistry Laboratory 1
Academic year:

2021
Views:

177

Pages:

20
Author:

Callboyx26e

PRELAB DISCUSSION #1 In the fields of observation, chance favors only the prepared mind. Louis Pasteur, University of Lille, Dec 7, 1854 Buffers and pH in Biochemistry How many parts are there to your investigations this week? PART I: Calibrating and using pH meters PART II: Making Tris Buffer pH 8 using two different methods PART II: Proteolytic Hydrolysis of BSA by trypsin via pHstat experiment This week you are expected to create your own data tables for all experiments as part of your prelab work. Make sure to take notes in the discussion to help you plan out your notebooks. Review of Part I & II- Procedure pH Meters How a pH electrode works Common Buffers in Biochemistry Labs • Buffers are solutions that resist changes in pH as acid and base are added and consist of a weak acid and its conjugate base • Buffers can only be used reliably within a pH unit of their pKa . HOW DO WE DECIDE WHICH BUFFER TO USE IN OUR EXPERIMENT? Physical Properties of Selected Buffers BUFFER Bis-Tris HEPES MES NaAc Tricine Tris UB1a UB2b UB3c UB4d pKa at 25 °C 6.46 7.55 6.15 4.75 8.05 8.06 Buffer Range 3.0-9.0 3.5-9.2 2.0-8.2 2.0-8.2 dpKa/°C at pH 7.0 N/A -0.014 -0.011 Negligible1 -0.021 -0.028 -0.015 -0.020 -0.012 -0.012 MW (g/mol) 209.24 238.3 195.2 82.03 179.2 121.14 Metal Binding Negligible1 Negligible1 Negligible1 Negligible1 Ca2+, Mg2+, Mn2+, Cu2+ Negligible1 Ca2+, Mg2+, Mn2+, Cu2+ Negligible1 Negligible1 Buffers and pH in Biochemistry How many parts are there to your investigations this week? PART I: Calibrating and using pH meters PART II: Making Tris Buffer pH 8 using two different methods PART II: Proteolytic Hydrolysis of BSA by trypsin via pHstat experiment Hydrolysis of a Peptide Bond What are the average pKa values for these functional groups? TOP HAT QUESTION 1 Trypsin is an enzyme that hydrolizes the peptide bond at the: a. b. c. d. C-terminal of (-) charged side chains C-terminal of (+) charged side chains C-terminal of hydrophobic side chains C-terminal of small side chains Table of α-Amino Acids Found in Proteins https://themedicalbiochemistrypage.org/amino-acids.php Each cleavage sets up two separate equilibria pH = pKa + log10 [A-] ------[HA] TOP HAT QUESTION 2 The pH of the solution is expected to _____ as your BSA is digested with trypsin starting at pH 8.5. a. increase b. decrease c. stay the same Food for Thought Considering the range of typical pKa values for the N- and C- terminal functional groups in proteins, do you expect to be adding base or acid to a proteolysis reaction mixture to maintain a physiological pH during the course of the reaction? WHAT DO YOU KNOW ABOUT YOUR STARTING MATERIAL, BSA? Amino Acid Sequence of BSA (in one-letter code): MKWVTFISLLLLFSSAYSRGVFRRDTHKSEIAHRFKDLGEEHFKGLVLIAFSQYLQQCPFD EHVKLVNELTEFAKTCVADESHAGCEKSLHTLFGDELCKVASLRETYGDMADCCEKQEPER NECFLSHKDDSPDLPKLKPDPNTLCDEFKADEKKFWGKYLYEIARRHPYFYAPELLYYANK YNGVFQECCQAEDKGACLLPKIETMREKVLASSARQRLRCASIQKFGERALKAWSVARLSQ KFPKAEFVEVTKLVTDLTKVHKECCHGDLLECADDRADLAKYICDNQDTISSKLKECCDKP LLEKSHCIAEVEKDAIPENLPPLTADFAEDKDVCKNYQEAKDAFLGSFLYEYSRRHPEYAV SVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKLGEYGFQ NALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHE KTPVSEKVTKCCTESLVNRRPCFSALTPDETYVPKAFDEKLFTFHADICTLPDTEKQIKKQ TALVELLKHKPKATEEQLKTVMENFVAFVDKCCAADDKEACFAVEGPKLVVSTQTALA The molecular weight of is 69293 Da and it contains 607 amino acids of which 60 are lysines and 26 are arginine residues. Review of Part III- Procedure Why are we using KOH rather than NaOH in this part of the experiment? Relating Titration to the # of Arg/Lys Residues Please note this is an example so you need to decide what you should be using for pH and pKa values in the HH equation for your own experiment. 2.5 9.5 pH = pKa + log10 [A-] ------[HA] [COO-] For carboxy- group: 8.5 = 2.5 + log10 -------  essentially all [COO-] [COOH] For amino- group: 8.5 = 9.5 + log10 [NH2] -------  [NH+3] [NH2] 1 -------- = ----[NH+3] 10 For every 11 bonds cleaved 11 H+ will form (from first equilibrium), but 1 will be used up in the second equilibrium and 10 will be left to be neutralized by KOH. So # of moles of KOH titrated x (11/10) = moles of peptide bonds cleaved. Relating Titration to the # of Arg/Lys Residues # of moles of KOH titrated x (11/10) = moles of peptide bonds cleaved Is this a universal formula? Total # of Lys+Arg per molecule of BSA= (moles of peptide bonds cleaved)/(moles of BSA used) Calculate moles of BSA using MW (66,000 g/mol) How much BSA did we use? 20 mls of 10 mg/ml solution = 200 mgs of BSA Substitute all values into the equation to determine total # of K+R WHAT CAN YOU DO EXPERIMENTALLY TO COLLECT THE BEST POSSIBLE DATA SET? WHAT CAN YOU DO DURING DATA ANALYIS TO GET THE MOST ACCURATE RESULTS? Always include in your work!!! • Clear mathematical or scientific basis for your work and what each number, line etc. represents • UNITS • Reasonable sig figs

Buffers and pH in Biochemistry

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