measuring ion binding of proteins in aqueous media l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Measuring Ion Binding of Proteins in Aqueous Media PowerPoint Presentation
Download Presentation
Measuring Ion Binding of Proteins in Aqueous Media

Loading in 2 Seconds...

play fullscreen
1 / 21

Measuring Ion Binding of Proteins in Aqueous Media - PowerPoint PPT Presentation


  • 196 Views
  • Uploaded on

VGJR-The B2K Group | BIOTRANSPORT & BIOREACTION KINETICS. Measuring Ion Binding of Proteins in Aqueous Media. Shelby Bickford Dr. V. G. J. Rodgers Department of Bioengineering University of California, Riverside. Introduction.

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Measuring Ion Binding of Proteins in Aqueous Media' - amelie


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
measuring ion binding of proteins in aqueous media

VGJR-The B2K Group| BIOTRANSPORT & BIOREACTION KINETICS

Measuring Ion Binding of Proteins in Aqueous Media

Shelby Bickford

Dr. V. G. J. Rodgers

Department of Bioengineering

University of California, Riverside

introduction
Introduction
  • This study will describe ion binding as a function of varying pH, ionic strength, and protein concentration.
  • The research will also determine the level of ionic concentration necessary for ion binding of proteins to become saturated.
  • This data will be gathered for the binding of sodium and chloride ions by four globular proteins.
background
Background
  • Ion binding of proteins has been studied for ~100 years.

Example: Klotz I, Walker M, Pivan R The Binding of Organic Ions by Proteins. 1946

  • Ion binding of proteins is a vital biological activity.

Example: Oxygen binding of Hemoglobin

  • A better understanding of ion binding of proteins has practical applications in many fields.

Example: Yamaguchi A, Tamang DG, Saier MHMercury transport in bacteria

http://www.psc.edu/~deerfiel/NIH/Protein-Calcium_Ion-Site_Thumb.gif

background4
Background

Hen Egg Lysozyme (HEL) – can break down bacterial cell walls and therefore help white blood cells engulf bacteria.

Bovine Serum Albumin (BSA) – responsible for shuttling fatty acids, and other water insoluble molecules through the body.

www.strubi.ox.ac.uk/.../crystallography.html

mgl.scripps.edu/.../pdb/pdb37/pdb37_1.html

Bovine Cytochrome-c – an electron donor in the electron transfer chain of the mitochondria. It also aids in apoptosis.

Bovine Alpha-Lactalbumin – an important whey protein found in cow’s, and other mammals, milk.

www.bath.ac.uk/~bsssi/ravi/lactal2.html

www.bio.davidson.edu/.../yeastproteins.html

background5
Background
  • Salt is naturally present in the body.
  • It helps maintain the electrolyte balance inside and outside the cells.
  • Knowing the effect naturally occurring salt has on protein binding is advantageous.
techniques for measuring ion binding
Techniques for Measuring Ion Binding
  • Dialysis Method – measures ion binding by either conductivity, difference UV spectroscopy, or collodion membrane electrodes.

www.kbac-hi.org/BMPs/content/def_conductivity.htmwww.chemicool.com/definition/spectronic_20.html

http://www.science.uwaterloo.ca/~cchieh/cact/c123/emf.html

  • Electromotive Force

Cell Method

previous studies
Previous Studies
  • Irving M. Klotz, F. Marian Walker, and Rita B. Pivan

The Binding of Organic Ions by Proteins. 1946

  • G. Scatchard, I. H. Scheinberg, and S. H. Armstrong, Jr.

Physical Chemistry of Protein Solutions. IV.

The Combination of Human Serum Albumin

with Chloride Ion. 1950

previous studies8
Previous Studies

C. Carr Studies on the Binding of Small Ions in

Protein Solutions with the Use of Membrane

Electrodes. III. The Binding of Chloride Ions

in Solutions of Various Proteins. 1953

W. Harris, A. Cafferty, S. Abdollahi,

And K. Trankler Binding of Monovalent Anions to

Human Serum Transferrin. 1997

methods and materials
Methods and Materials
  • Dialysis Method using conductivity as a measure of ionic strength.
  • 0.5 ml of 0.001M BSA solution was injected into the dialysis cassette.
  • 3 cassettes were placed into a water-tight bag containing 3 ml of NaCl solution: 0.15 M, 0.1 M, 0.01 M, or 0.001 M.
  • Bags were used as opposed to beakers so that a low volume of solution could be used.
methods and materials10
Methods and Materials
  • Each NaCl solution is assessed with an Orion conductivity probe, an Orion Chloride Ion Selective Electrode (ISE), and a pH meter before the cassettes are added.
  • Once equilibrium is reached the solution is analyzed again.
  • The difference in conductivity allows the number of bound ions to be calculated.

Orion ISE Probe

Orion pH Meter

Orion Conductivity Probe

conductivity calculations for nacl
Conductivity Calculations for NaCl

Concentration

Conductivity

at a

Theoretical Values of conductivity given in

temperature of 25° C. Concentration is in mol/L.

electroneutrality

Solution Chamber [ I ]

Solution Chamber [ II ]

M+

X-

M+

X-

Pn+

OH-

H+

H+

OH-

Electroneutrality

Electrical Neutrality:

Ion Activity Equilibrium:

Picture courtesy of Yiheng Wang

slide15
Data

Test #3

NaCl solution = 0.001M

BSA solution = 0.001M

Initial conductivity = 119.9 μS/cm

ISE reading = 0.0009M

pH = 6.9

Conductivity Temperature

in μS/cm in °C

114.9 26.2

113.4 26.0

112.3 24.2

103.3 24.3

97.3 23.4

79.4 26.0

71.7 29.8

Overall change = 48.2 μS/cm

Test #4

NaCl solution = 0.01M

BSA solution = 0.001M

Initial conductivity = 1066 μS/cm

ISE reading = 0.0081M

pH = 6.431

Conductivity Temperature

in μS/cm in °C

804 26.7

655 25.4

446 23.3

246.9 26.7

230.9 25.1

Overall change = 835.1 μS/cm

data cont
Data (cont.)

Test #5

NaCl solution = 0.1M

BSA solution = 0.001M

Initial conductivity = 10.17 mS/cm

ISE reading = 0.0736M

pH = 7.159

Conductivity Temperature

in mS/cm in °C

5.27 23.0

3.03 28.7

1.194 27.0

Overall change = 8.976 mS/cm

discussion
Discussion
  • Volume loss affecting conductivity readings
  • Better accuracy with larger volumes
future objectives
Future Objectives
  • Improve experimental model to increase accuracy
  • Measure ion binding in high concentration protein solutions
  • Control pH to determine its affect on ion binding
slide19

Acknowledgements

  • Thanks to Dr. Victor Rodgers & the B2K Group, UCR, Denise Sanders, Jill Brady, and the BRITE program.

Questions?

Shelby Bickford at

shelby.fawnb@gmail.com

goals and questions
Goals and Questions
  • Ion binding as a function of:

- Ionic concentration

- pH

-Protein concentration

  • Electroneutrality condition
  • Ability to measure each species of ion
donnan equilibrium and electroneutrality
Donnan Equilibrium and Electroneutrality
  • The albumin molecule is not uniformly charged within the primary structure. At neutral pH, Peters (1985) calculated a net charge of -10, -8, and 0 for domains I, II, and III for bovine serum albumin. http://www.friedli.com/research/PhD/chapter5a.html
  • Since the BSA molecule is negatively charged, and is said to bind mostly anions, how can electroneutrality be satisfied? If cations and anions are in a 1:1 ratio, how will there ever be enough cations to balance both systems?
  • www.nature.com/.../n6994/fig_tab/429822a_F1.html