Anfinsen's experiment

1. Anfinsen’s experiment
Presented by-
Shubhangi Singh
Roll no. 201648
MSc. Biotechnology
Central university of Haryana

2. Christian Boehmer Anfinsen Jr. (March 26, 1916 – May
14, 1995) was an American biochemist. He shared the
1972 Nobel Prize in Chemistry with Stanford
Moore and William Howard Stein for work
on ribonuclease.
Christian B. Anfinsen, NIH portrait, 1969 - Christian
B. Anfinsen - Wikipedia
What is the basis of the catalytic power of
enzymes?

3. Why RNase A?
Insulin was the first protein whose amino acid sequence was known No convenient activity assay
Ribonuclease A
Assayed by its ability to degrade yeast nucleic acid
Available in large quantities, cheap, from slaughterhouses
Stable during purification
Description of Bovine Pancreatic Ribonuclease A: • 124 amino acids
• 13,700 Daltons
Bovine Ribonuclease (primordion.com)

4. A loss of three-dimensional structure
sufficient to cause loss of function is
called protein denaturation.
Protein structure: Primary, secondary, tertiary &
quatrenary (article) | Khan Academy
Denaturation of protein - Bioscience Notes
Denaturation of proteins
Top 30 Protein Structure GIFs | Find the best GIF on Gfycat
Top 30 Protein Structure GIFs | Find the best GIF on Gfycat

5. Sulfhydryl group pairings in
native conformation
58-110
72-65
26-84
40-95
Two cysteines in proximity can
form a covalent bond called
disulfide linkage.
SH groups are oxidized to form
disulfide bonds.
Native state of ribonuclease A

6. .
RNase A - Proteopedia, life in 3D

7. 2-MERCAPTOETHANOL
2-mercaptoethanol cleaves the disulfide
bonds that may form between thiol groups
of cysteine residues.
Urea
Urea interacts with the polar regions of the
proteins resulting in the unfolding of the
tertiary and secondary structure of the
proteins and exposure of the hydrophobic
core to water and urea, simply put- urea
denatures proteins
Proteins - Biomolecules Chemistry Notes | EduRev

8. The refolding ribonuclease A after full denaturation by
reductive cleavage of its four disulfide bonds required that
only one of the 105 possible pairings of eight sulfhydryl
groups to form four disulfide linkages take place.
7 5 3 1 105
1/105 = NATIVE
CONFORMATION
Nobelprize.org. n.d. [online] Available at: <https://www.nobelprize.org/uploads/2018/06/anfinsen-
lecture.pdf> [Accessed 2 December 2021].
Lehninger Principles of Biochemistry, 8th Edition

9. RNase A was treated with 8 M urea and 2-mercaptoethanol
which resulted in the complete unfolding of the enzyme and
the reductive cleavage of all the 4 disulfide bonds
Fundamentals of Biochemistry Life at the Molecular Level by Donald Voet, Charlotte W. Pratt, Judith G. Voet (z-lib.org).pdf
Anfinsen’s experiment

10. Removal of both urea and 2-mercaptoethanol and exposure
to excess oxygen resulted in reformation of the disulfide
linkages resulting in protein with 100% enzymatic activity. The
resulting protein was physically indistinguishable from the
native enzyme.
Fundamentals of Biochemistry Life at the Molecular Level by Donald Voet, Charlotte W. Pratt, Judith G. Voet (z-lib.org).pdf

11. If disulfide linkage formation was random
Probability of getting the native conformation= 1/105
But, 100% enzymatic activity and physically
indistinguishable
=> Under renaturation conditions, disulfide linkage is
not randomly formed, and are indicated to be dictated
by a template

12. When RNase A was oxidized in the presence of urea, protein mixture only has ~1% of the native enzyme activity.
Indicates, under denaturating conditions disulfide linkage formation is random.
Removal of urea after the disulfide bonds are formed results in the formation of a scrambled protein.
Fundamentals of Biochemistry Life at the Molecular Level by Donald Voet, Charlotte W. Pratt, Judith G. Voet (z-lib.org).pdf

13. This “scrambled” protein can be made fully active by exposing
it to a trace of 2-mercaptoethanol, which breaks the improper
disulfide bonds and allows the proper bonds to form.
Fundamentals of Biochemistry Life at the Molecular Level by Donald Voet, Charlotte W. Pratt, Judith G. Voet (z-lib.org).pdf

14. Fundamentals of Biochemistry Life at the Molecular Level by Donald Voet, Charlotte W. Pratt, Judith G. Voet (z-lib.org).pdf

15. Anfinsen’s work demonstrated that proteins can fold spontaneously
into their native conformations under physiological conditions. This
implies that a protein’s primary structure dictates its three
dimensional structure.

16. Adaptation of Anfinsen's Experiment
An Adaptation of Anfinsen's Protein-Folding Experiment for Classroom Investigation | The American
Biology Teacher | University of California Press (ucpress.edu)
The clear zone at indicates
that RNase had diffused and
broken down the RNA around
the well
At B, no clear zone has
formed, showing that
RNase had been
denatured by 2-ME and
urea by being unfolded
into polypeptide chains
At C, when the denaturants had
been removed by dialysis, a
smaller clear zone was
observed. This shows that some
RNase molecules had regained
catalytic activity by refolding
back into its native, three-
dimensional shape
spontaneously.
The clear zone at D is
as large as that at A,
indicating that
boiling had no effect
on the activity of
RNase because its
four disulfide bonds
strongly hold the
conformation of the
molecule.
The breakdown of RNA is not
due to the buffer and
denaturants, as shown by the
absence of clear zone at E.
The amino acid sequence of a
protein contains all the information
required for three dimensional
structure of the protein

17. References
Nobelprize.org. n.d. [online] Available at: <https://www.nobelprize.org/uploads/2018/06/anfinsen-lecture.pdf>
[Accessed 2 December 2021].
Lehninger Principles of Biochemistry, 8th Edition | Macmillan Learning for Instructors. [online] Available at:
<https://www.macmillanlearning.com/college/us/product/Lehninger-Principles-of-Biochemistry/p/1319228003>
[Accessed 2 December 2021].
Voet, D., Voet, J. and Pratt, C., 2016. Fundamentals of Biochemistry. New York: Wiley.
An Adaptation of Anfinsen's Protein-Folding Experiment for Classroom Investigation | The American Biology Teacher | University
of California Press (ucpress.edu)