This document describes an experiment investigating the fibrillation kinetics of bovine and human insulin fragments and a tailored peptide. Key findings include: - Bovine insulin fragments aggregated faster than human fragments under Thioflavin T fluorescence spectroscopy, indicating higher fibrillation propensity. - Higher temperature (60°C) and lower pH (2.5) increased fibril formation compared to lower temperature (40°C) and higher pH (7.0). - The tailored diblock peptide showed no fibril formation over 4 weeks under the experimental conditions.

1. Results
Insulin ThT
• Figures 3 and 4 show the fibrillation results for
human and bovine insulin respectively.
Materials and Methods
Peptide Synthesis
• Sequence of human fragment: Asn-Glu-Leu-Gln-
Tyr-Leu-Ser-Cys-Ile-Ser-Thr
• Sequence of bovine fragment: Asn-Glu-Leu-Gln-
Tyr-Leu-Ser-Cys-Val-Ser-Ala
• Sequence for tailored peptide: Leu-Leu-Leu-Leu-
Leu-Lys-Lys-Lys-Lys-Lys
• Each peptide has a lysine tail in an attempt to
increase its solubility
Introduction
Diseases such as Alzheimer’s and Parkinson’s are
linked to amyloid deposits, which are insoluble
protein aggregates. Some polypeptides that form
amyloid fibrils are insulin and (Lys)n-b-(Ile)m. We
found that the fibrillation of diblock peptides is
around 72 hours from Naik (2012) at pH 4. The
diblock peptide used here can be shown in Figure 1.
We also found that bovine and human insulin have
different fibrillation kinetics in spite of the fact of
being almost identical molecules; they differ in four
amino acids (Figure 2). We hypothesized that
shorter fragments of insulin variants that include the
region which lacks homology should have the same
differences in the kinetics of fibrillation. Thioflavin-T
Fluorescent Spectroscopy (ThT) was used to explore
the differences.
Material and Methods Continued
Experiment of Design
Fibrillation of Bovine and Human Insulin Fragments and Tailored Peptides
Al-Lozi, A., Snyder, A., and Dr. Forciniti
Chemical and Biochemical Engineering
Missouri University of Science and Technology
Conclusion
• Fragments of bovine and human insulin seem
to be forming amyloid fibrils
• It is not clear if there is a kinetic difference
between the two fragments
• ThT results suggest that the bovine fragment
has a higher propensity to form fibrils
• The pH’s used in this research were different
from Naik, therefore, no conclusions can be
made on the fibrillation of diblock peptides
Results Continued
Thank you Dr. Daniel Forciniti for your assistance and OURE at Missouri S&T for funding
Materials and Methods
Fibrillation
• 17 solutions containing 10mg and 20mg of
each insulin fragment (human and bovine) in 1
mL of 2mM sodium phosphate buffer, pH 2.5
and pH 7
• 24 solutions containing 10mg and 20mg of the
tailored peptide in 1mL of 2mM sodium
phosphate buffer, pH 2.5 and pH 7
• The samples were then incubated at 60°C (with
and without agitation) and at 40°C (with and
without agitation)
• Two samples of bovine insulin were started on
March 1, 2015 to test the conditions for the
run that should produce the most fibrils and
the run that should produce the least amount
of fibrils
• The next set of samples were started March 20,
2015 for both the 17 insulin samples and 24
tailored peptide samples
ThT (Thioflavin-T Fluorescence Spectroscopy)
• A Stock solution was prepared by adding 4 mg
of Thioflavin T to 5 mL of phosphate buffer
• 20µL were added to every 1mL sample. More
was added every two weeks due to the
degradation of the ThT working solution
• The excitation wavelength was fixed at 480nm
with extra wavelength set at 490nm and
500nm
• The samples were tested once every week in
triplicate
From the graphs, much information can be
viewed:
• There is a visible difference between the
bovine and human fibrillation as the bovine
insulin aggregates faster than the human
insulin
• There is also a difference between the
temperatures and pH levels where the higher
temperature and lower pH was more likely to
produce more fibrils than the lower
temperature and higher pH
• As the incubation time increases, the
concentration of fibrils increase as well
Tailored Peptide ThT
• Fibrils were expected to form within the first
week for the diblock peptides. However, this
was not the case. At first, it appeared that the
fibrils exhibited slow kinetics under the
conditions of the experiment. The samples
were left to incubate for a total of four weeks,
and no sign of fibrils were shown under ThT
fluorescence.
Ala
ValAla
Leu Leu Leu Leu Leu Lys Lys Lys Lys Lys
Diblock Peptide
Figure 1: Amino acid sequence of tailored peptide of diblock
Figure 2: Amino acid sequence of human insulin and the difference of Bovine insulin
0 200 400 600 800 1000 1200 1400 1600
Week 1
Week 2
RFU @ 480
Time(weeks)
Run 12 Run 8 Run 6 Run 5 Run 3 Run 13 Run 11 Run 10 Run 9 Run 7 Run 4 Run 2 Run 1
Figure 3: Human insulin data for two weeks after initial incubation
Figure 4: Bovine insulin data from initial incubation
Run pH Temp. (°C) Agitation Concentration (mg/ mL)
1 2.5 40 off 10
2 2.5 40 off 20
3 2.5 40 on 10
4 2.5 40 on 20
5 2.5 60 off 10
6 2.5 60 off 20
7 2.5 60 on 10
8 2.5 60 on 20
9 7 40 off 20
10 7 40 on 10
11 7 60 off 10
12 7 60 on 20
13 7 40 off 10
Table 1: Experimental Design for Human and Bovine insulin fragments and tailored peptide incubation.
0
1000
2000
3000
4000
5000
6000
7000
Week 0 Week 1 Week 2
RFU@480
Time (weeks)
Run 1 Run 2 Run 12 Run 11 1 2 11 12