Effect of pH and Ionic Strength on Solubility of Proteins

Slides:



Advertisements
Similar presentations
Non-redox Reactions/ Double Replacement Reactions
Advertisements

Electrolytes Some solutes can dissociate into ions. Electric charge can be carried.
simple protein hydrolyze to yield only amino acids e.g.: albumins, globulins. conjugated protein is a protein that functions in interaction with other.
Isolation of Casein from Milk
Effect of pH and Ionic Strength on Solubility of Proteins
Chapter 15 Solutions. Chapter 15 Table of Contents Copyright © Cengage Learning. All rights reserved Solubility 15.2 Solution Composition: An Introduction.
Pre-Lab 4.1. Purpose To determine if you can dissolve all of a solid into a liquid.
Salting in and Salting out of proteins and Dialysis (Isolation Of Lactate Dehydrogenase Enzyme ) BCH 333 [practical]
Lab Activity 7 Proteins Part I IUG, 2015 Dr. Tarek Zaida 1.
Acid-Base Equilibria and Solubility Equilibria Chapter
Ions in Aqueous Solutions and Colligative Properties
Acids, Bases, pH and Buffers. Acids An acid dissolves in water to donate H + In water the H+ reacts with water to make the hydronium ion H + + H 2 O 
Lipids.
Lab 27 - Determination of K sp for a Sparingly Soluble Salt Introduction Inorganic substances are broadly classified as acids, bases, or salts. With few.
Precipitation of Proteins at isoelectric Point A.Protein solubility: The solubility of proteins in aqueous buffers depends on the distribution of hydrophilic.
Mixtures and Solutions
Isolation of casein from milk
Qualitative test of protein
Solutions!. What is a solution? A homogeneous mixture! Made up of a solute and solvent.
Faculty of Medicine Biochemistry Department Practical Biochemistry Precipitation of Proteins A/Prof. Magdy Elnashar (Preparatory Year)
Chemical Calculations for Solutions (Ch 12) Dr. Harris Lecture 12 Suggested HW: Ch 12: 1, 10, 15, 21, 53, 67, 81.
Solutions CH 13. Two Types of Mixtures Homogeneous Same throughout, looks pure EX: Air Heterogeneous Different throughout EX: Sand.
Writing a Lab Report. Scientific Method When performing a lab experiment we follow the scientific method. What is the scientific method? Purpose, Hypothesis,
Review of Chemistry 11. Ionic Compounds:Covalent Compounds: (Begins with a metal or NH 4 ) (Begins with a nonmetal) BaseSalt AcidNonacid NaOH (Metal +
Option # 1. Key Points ► Law of Conservation of Mass -  Mater is neither created nor destroyed during a chemical reaction. ► Solubility –The max amount.
Chapter 18 The Solubility Product Constant. Review Quiz Nuclear Chemistry Thermochemistry –Hess’s Law –Heats (Enthalpies) of…
EXPERIMENT: Action of Amylase on Starch. A B C D E F G Add 10 ml of distilled water to each tube.
Forming Solutions 15.1: Pgs Objectives To understand and describe the process of dissolving To understand and describe the process of dissolving.
Protein purification always begin with intact tissue  Disrupt  Blender, homogenizer  Remove debris  Centrifugation  Precipitate/concentrate  Ammonium.
Chapter 15: Solutions 15.1 Solubility 15.2 Solution Composition 15.3 Mass Percent 15.4 Molarity 15.7 Neutralization Reactions.
Section 6.2—Concentration
Lab Activity 9 Precipitation Of Proteins
Solutions. Definitions Solution – Homogeneous mixture of two or more substances Solute – Substance that is dissolved Solvent – Substance that dissolves.
Acid-Base Reactions Ch. 15. Acid-Base Reactions Neutralization reactions Neutralization reactions – pH is changed Produce a salt and H 2 O Produce a salt.
Separation of a Mixture
BUFFERS SUROVIEC SPRING 2014 Chapter I. Buffer Solutions A. Buffer is a solution that resists a change in pH with the addition of small amounts.
Titrations. Standard Solution Sample Solution Burette A titration is a volumetric analysis technique used to find the [unknown] of a sample solution by.
Whole milk contains vitamins (vitamins A, D, and K), minerals (calcium, potassium, sodium, phosphorus,), proteins (which include all the essential.
Making Salts Insoluble salts – precipitation Mix two solutions together one with the cation, one with the anion. Filter, wash and dry. Soluble Na +, K.
Section 15.1 Forming Solutions Steven S. Zumdahl Susan A. Zumdahl Donald J. DeCoste Gretchen M. Adams University of Illinois at Urbana-Champaign Chapter.
Lab 6 Ig Purification What will the Ig be used for? How pure does it need to be? What is the source of the antibody (serum, ascites, cell culture supernatant)
Experiment (2) Determination of the Heat of Neutralization of Strong Acid with a Strong Base.
Separation of Casein.
Unit X - Solutions Chapter Goals 1. Understand the process of dissolving and why certain substances dissolve in water. 2. Understand the qualitative.
1 Chapter 4 Aqueous solutions Types of reactions.
Lab Activity 11 Purification of LDH Part II
Lab Activity 12 Purification of LDH Part II
Chemical Changes and Structure
Isolation of casein from milk
Salting in and salting out of proteins and dialysis
Particles in Solution.
BCH 447- Metabolism: Mid term date: Mon 7/2/ /11/2017
Isolation of casein from milk
Chapter 6: Solutions.
DO NOW Get out notes and homework handout..
What is a Solution? Solution – homogeneous mixture
Qualitative tests of protein
Isolation of casein from milk
Lab Activity 7 Proteins Part I
Controlling Variables
Experiment 7.
Organic solvent extraction
Biochemical Methodology
Expt A The Grignard Synthesis of Triphenylmethanol
(Chemists have Solutions!)
Lipids-I.
Gravimetric Analysis.
EXP. NO. 6 Acid Base Titration
Lab Activity 11 Purification of LDH Part II
Isolation of casein from milk
Presentation transcript:

Effect of pH and Ionic Strength on Solubility of Proteins Milk Proteins Effect of pH and Ionic Strength on Solubility of Proteins

INTRODUCTION Food Industry: Examples: - Functional Properties - Nutritional Gelation Foaming Change in viscosity Examples: Whole eggs, egg yolk, egg albumen, whey solids, non-fat dry milk

Types of Proteins Isolates: 90-95% protein by weight Concentrates: 50-70% Methods: Differences in solubility – function of pH and ionic strength of environment Purification – size exclusion techniques (ultrafiltration) Chromatographic approaches - particle size - density - Charges - polarity

Influences of pH on solubility Due to overall charges: - NH2 COOH Groups on side chain In order words: Positively charged: ------------ At Low pH Negatively charged: ------------- At high pH ISOELECTRIC POINT OF A PROTEIN: Intermediate pH at which the net charge zero

Influences of pH on solubility Interaction with water: Either positive or negatively charged Soluble At isoelectric point: ???

Milk Proteins Isoelectric point of Casein Remaining proteins: May be precipitated with salts

“Salting out” Salting Out: Proteins have unique solubility profiles in neutral salt solutions. Low concentrations of neutral salts may: Increase the solubility of some proteins Precipitate from solution as ionic strength is increased. Actions are somewhat unique to each protein. Ammonium sulfate (NH4)2SO4 is commonly used because it is highly soluble and very effective. NaCl or KCl may be also be used to “salt out” proteins.

“Salting out” Ionic Strength = ½ S MiZi2 Mi = Molarity of ion Zi = Charge of ion 1M NaCl = ½ S (1 X 12) + (1 X 12)= 1 1M CaCl2 = ½ S (1 X 22) + (2 X 12) = 3 1M (NH4)2SO4 = ½ S (2 X 12) + (1 X 22) = 3 Na+ Cl- Ca2+ Cl- NH4+ SO42-

Objectives of Lab: To illustrate the effects of pH on protein solubility. To provide a better understanding of the isoelectric point of a protein. To examine the influence of salt addition on protein solubility.

Procedure - Casein 1. 2. 3. 4. Casein Whey (soluble) Heat to 40°C pH 4.6 (1.0M HCl) 1. Weigh 50mL of Skim milk Heat to 40°C Adjust pH with 1.0M HCl (to 4.4 to 4.6) 2. Weigh a 50mL falcon tube – Register Separated Casein from whey: add to falcon tube Centrifuge it for 10 minutes 3. Pour whey into a beaker: Save it!!! Retain insoluble proteins in tube. 4. Casein: Re-weigh solids in tube. Add to original beaker: Calculate amount of hydrated casein Centrifuge 10 Min Casein Whey (soluble) Weigh Calculate hydrated casein

Procedure 5. 6. Add 50mL to casein curds Suspend the casein curd in 50mL of water. SLOWLY and DROP WISE add 1.0M NaOH until pH 7.5, STIR CONSTANTLY 6. Register observations pH 7.5 1.0 NaOH CONTINUOUS STIR!!!!

Discuss your observations Procedure - Whey 7. Take Whey proteins (pH 4.6, approximately 50mL) Weigh 30g Ammoniun sulfate Add half NH4SO4 slowly to Whey– with magnetic stirrer constant stirr for 5min: To salt out of proteins 8. Weigh falcon tube (50mL) Add whey protein and centrifuge for 5min Shake well and add whey back to beaker 9. Slowly add the rest of the Ammonium sulfate to the whey (stirring for 5min) and repeat centrifugation Pour off supernatant and weigh the residual protein precipitate. Add ½ NH4SO4 SLOWLY Centrifuge 5 Min Supernatant Discuss your observations

Results: Calculate the protein yield of casein and whey proteins and compare your yields with values reported in the literature. Do your values agree or disagree with those in the literature? Why or why not (keep in mind that you have hydrated proteins, not dried proteins). Define the concept of “isoelectric point”. According to your results, what is the isoelectric point of casein? What happened to casein at higher pHs? What is “salting out”?, why did we choose to “salt out” the whey proteins instead of just adjusting the pH?

Discussion: Project You just took a job with Protein USA, a major supplier of all forms of protein to the food industry. Your supervisor assigns you to a project for Nestle USA, one of the world’s largest food companies, and their most important customer. Nestle is developing a new variety of Lean Cuisine entrees, targeted to vegetarian consumers, and are interested on using soy as the major protein source and are thinking about buying from your company. However, your Nestle customers want to know more about the basic principles behind the soy protein isolation process, the major functional properties of your protein isolate, and their potential applications (eg. meat replacers, textured foods).