Enhanced amyloid fibril formation of insulin in contact with catalytic hydrophobic surfaces

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Institutionen för fysik, kemi och biologi

Examensarbete

Enhanced amyloid fibril formation of insulin in contact

with catalytic hydrophobic surfaces

Belma Salagic

Examensarbetet utfört vid Linköpings Universitet

Linköping 2007

LITH-IFM-EX--07/1789--SE

Linköpings universitet Institutionen för fysik, kemi och biologi 581 83 Linköping

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Institutionen för fysik, kemi och biologi

Enhanced amyloid fibril formation of insulin in contact

with catalytic hydrophobic surfaces

Belma Salagic

Examensarbetet utfört vid Linköpings Universitet

Linköping 2007

Per Hammarström, Malik M. Ali

IFM, Linköpings Universitet

Examinator

Per Hammarström

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URL för elektronisk version

ISBN

ISRN: LITH-IFM-EX--07/1789--SE

_________________________________________________________________

Serietitel och serienummer ISSN

Title of series, numbering ______________________________ Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Titel Title

Enhanced amyloid fibril formation of insulin in contact with catalytic hydrophobic surfaces

Författare Author

Belma Salagic

Nyckelord Keyword

Insulin fibrillation, Amyloid fibrillation, Catalytic hydrophobic surfaces

Sammanfattning Abstract

The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems since huge volumes of the product go to waste.

In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these kinds of conditions such as high

temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster.

In the following experiment I am going to investigate if the experiments and conclusions done before, where different kinds of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal

fibrillation under the same conditions. Avdelning, institution

Division, Department

Chemistry

Department of Physics, Chemistry and Biology Linköping University

Datum

Date

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Abstract

The important protein hormone insulin, responsible for different kind of functions in our body but mainly storage of nutrients, has for a long time been used for treatment of diabetic

patients. This important protein is both physically and chemically unstable. Especially during production where the insulin protein is exposed to unnatural environmental conditions such as acidic pH has this been causing problems, since huge volumes of the product go to waste. In the human body the environment for the protein is tolerable with normal body temperature and the right pH, but when the protein is commercially synthesised the environmental

conditions are not ultimate. What happens during these unfavourable conditions is that the insulin starts to fibrillate. Meaning that linear, biologically inactive aggregates are formed. If then under these conditions such as high temperature and acidic pH, the insulin comes in contact with hydrophobic surfaces then the fibrillation of the protein goes even faster.

In the following experiment I am going to investigate if the experiments and conclusions done before, where different kind of additives to insulin solutions have been used to enhance the amyloid fibrillation of insulin, are as effective as it has been proposed and I am going to prove that the presence of hydrophobic surfaces, such as coated silicon surfaces or glass and

addition of preformed fibrils, so called seeds, increase amyloid fibrillation of the insulin protein under certain conditions, in comparison with the normal fibrillation under the same conditions.

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Content

1. Insulin molecule and amyloid fibril formation ...1

2. How and why the kinetic experiment was going to be done. Expected effects of seeds and surfaces ...2

3. What instruments were being used to study kinetics of insulin amyloid fibrillation ...3

4. Experimental procedures ...4

4.1. Experiment 1 ...5

4.1.1. Making the stock solution...5

4.1.2. Making the surfaces ...6

4.1.3. Kinetic experiment ...7

4.1.4. Fluorescence measurements...8

4.2. Experiment 2 ...13

4.2.1 Making the stock solution...13

4.2.2. Making the surfaces ...14

4.2.3. Kinetic experiment ...15

4.2.4. Fluorescence measurements...16

5. Difference between washed and not washed stained surface ...24

6. Discussion and source of error – what could have been done better?...25

7. References ...26 Appendix I ...27 Appendix II ...29 Appendix III...33 Appendix IV...35 Appendix V ...37 Appendix VI...39 Appendix VII ...41 Appendix VIII ...43

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1. Insulin molecule and amyloid fibril formation

The hormone insulin is a protein consisting of 51 amino acids, divided on two chains. One A – chain containing 21 amino acids and one B- chain containing 30 amino acids. It is produced in the pancreas and is catabolized in the liver, kidney and placenta. This important hormone is mainly promoting storage of ingested nutrients and fat but it is also responsible for many other functions, like promoting protein synthesis in the muscles.3

Effective treatment of diabetic patients with insulin have been taking place for a long time now, and constant improvement and optimization of both therapeutic use and production of insulin is therefore of the greatest importance.2

The integrity of insulin is strongly dependent on environmental conditions making it both physically and chemically unstable.1,2This means that simple changes like the pH, effects the state of the protein. In solution it exists in three states: hexameric, dimeric and monomeric and for example a small concentration of acid, such as HCl, makes the monomeric state predominant.1

A major stability problem is amyloid-like fibril formation, a process where linear, biologically inactive, aggregates are formed, by interactions between non native insulin molecules. In patients with type II diabetes amyloid deposits have been seen after repeated injections and in normal aging, but still the largest problem with aggregation of insulin is during production where large quantity of the insulin needs to be thrown away during commercial isolation and the purification steps, where many of them involve pH in the range of 1-3.2 Fibril formation consists of three reaction steps. The first one is nucleation, where several misfolded protein monomers form an organized structure so called the nucleus. This nucleus is a precursor for the second reaction. In the second reaction elongation of the nucleus is taking place, meaning more monomers are added and the nucleus is growing into fibrils. The last reaction is floccule formation or so called precipitation.2

Since the first step in fibril formation is dependent on a temperature above ambient it is possible to study the formation of fibrils as a kinetic experiment, taking samples of incubated insulin over time and comparing the amount of formed fibrils. By studying the kinetics of the fibril formation of insulin, researchers are hoping to reveal important information making it possible to prevent this process.

It has been proposed that the aggregation of insulin is hydrophobic in nature. That the initial step is formation of conformationally changed monomers where the hydrophobic face of the insulin molecule that is normally buried in the diamer and hexamer, becomes exposed to solvent. And it has been proved that insulin forms fibrils more easily in the presence of hydrophobic surfaces.2,4

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2. How and why the kinetic experiment was going to be done. Expected

effects of seeds and surfaces

To prove that different hydrophobic substances enhance the amyloidal fibrillation of the insulin protein, I studied the kinetics of fibrillation process of insulin at 60˚C, using different solutions of 200 µM bovine insulin diluted in 25 mM HCl giving me a solution with pH 1,6. These solutions differed from each other because, to some of the samples I added

hydrophobic coated silicon surfaces that I prepared by repeated incubation in insulin,

followed by surface drying before the kinetic experiment and to some samples I added small amount of so called seeds. Beside these two additives I also used glass surfaces prepared in the same way as the silicon to see if the effect would be the same.

According to previous research in presence of hydrophobic surfaces such as hydrophobic silicon surfaces or air, the fibrillation of insulin happens much more rapid then normally. If then the silicon surfaces have already been pre-incubated in insulin solution to have a thin layer of fibrils, the fibrillation goes even faster making the lag time that normally exists in the process, almost disappear. The thin surface of fibrils seams to be working as a catalyst.4 Even volume difference affect the fibrillation rate, probably since larger volume means more monomers for fibrillation.

Since the hydrophobic silicon surfaces have been proved to be very effective I tried another hydrophobic surface, glass, to see if the effect would be the same. The surfaces of glass is normally hydrophilic and by using the same procedure to prepare it as with the silicon surfaces where a hydrophobic coat is made on the surface this seamed to be an interesting alternative to the silicon since it is easier to later on investigate the surface with an optical microscope.

The seeds I am using are actually nothing else but already preformed fibrils in solution.2These have also been proved to speed up the fibrillation when added and even small amounts make big difference on the rate of fibrillation.4,2

The samples I am comparing are different incubated volumes of plain insulin stock solution, insulin solution with silicon surfaces, insulin solution with glass surface and insulin solution with 0,17% seeds. Each sample is stained with thioflavin, ThT and their fluorescence intensity is measured and plotted versus the incubation time making the comparison possible.

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3. What instruments were being used to study kinetics of insulin amyloid

fibrillation

The fibrillation kinetics was studied by measuring the fluorescence intensity of every solution over time. For these measurements the dye thioflavin, ThT was used.

ThT stained insulin fibrils have been shown to fluorescence more intensive by giving rise to a new excitation maximum at 450 nm and enhanced emission at 482 nm. The interaction of ThT with amyloid fibrils is believed to be specific and rapid where only multimeric fibril forms of the protein fluorescence. This makes it an effective tool for studying the insulin fibril formation kinetics.2

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4. Experimental procedures

Material:

Dry bovine insulin 25 mM HCl 2 M GuHCl Magnetic stirrer Automatic pipette Dialysis tube 0,45 µm filter

Spectrophotometer (two kinds of spectrophotometer, one for absorbance measurements and one for fluorescence measurements)

Tweezers

Silicon surfaces (ca.5x5mm) Glass surfaces (ca.5x5mm)

TL-1 (a mixture of 5 parts H2O, one part NH3 and one part H2O2)

TL-2 (a mixture of 6 parts H2O, one part HCl and one part H2O2)

DDS (1% dichlorodimetylsilane in xylene) Ellipsometer Ultrasonicater N2 Heating block Thermometer Vortex

Preformed insulin amyloid seeds Microtiter plate

ThT solution (4 µM ThT (thioflavin) in 50 mM phosphate buffer and 100 mM NaCl pH 7,5) Congo red dye

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4.1. Experiment 1

4.1.1. Making the stock solution

I started the experiment by making the stock solution of 200 µM bovine insulin. This solution was both used for the kinetics experiment and for coating the hydrophobic surfaces.

Since the insulin I was using was dry and freezed from the beginning I started with dissolving approximately 16 mg of the protein with 1,5 ml of 2 M GuHCl in an Nunc tube by vortex. When all the insulin was dissolved it had to be purified by dialysis against cold 25 mM HCl in 4˚C for at least eight hours under constant stirring. The reason why the solution has to be kept cold is because of insulin’s tendency to denature, since it is both physically and chemically unstable at acidic pH.

When the dialysis was done, I filtered the insulin with a 0,45 µm filter to a Nunc tube. This filter fit the insulin molecule, so that the larger particles and impurities are captured while the pure insulin will end up the Nunc tube.

To check the insulin concentration of the solution I had I measured the UV spectrum between 250 and 400 nm with a spectrophotometer and the peak at 277 nm was used.

Since the solution I had is known to have an approximately large concentration in comparison to the end concentration that I needed, I diluted a small amount required to measure the absorbance twenty times with cold 25 mM HCl. This made it easier to measure the spectra and to get a more correct value of the absorbance.

The absorbance I got at 277 nm was 0,299. To transform this to concentration I simply divided this number with the so called extinction coefficient of insulin protein and then multiplied it with the times the solution was diluted.

ABS_ x times diluted = concentration in M Ex.coeff

0,299 x 20 = 0,001 M = 1 mM 5747

Now that I knew the concentration of the solution I could dilute it to the concentration I needed that was 200 µM with cold 25 mM HCl.

C1V1 = C2V2

The finial volume I needed was 7 ml of 200 µM insulin solution. 1,0 mM x V1 = 0,2 mM x 7 ml Î

V1 = 1,4 ml

Of the total volume of 7 ml stock solution, 5,5 ml was stored in the refrigerator at 4˚C and 1,5 ml was going to be used for the preparation of 2 silicon and 1 glass surface.

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4.1.2. Making the surfaces

The next step of the experiment is making the hydrophobic surfaces.

I started with cutting two silicon and one glass surface ca. 5x5 mm with a diamond knife. To make sure that there were no impurities on them that could affect the result, the surfaces had to be carefully cleansed. This was done in two steps.

The first step was boiling the surfaces in a mixture consisting of 5 parts H2O, one part NH3

and one part H2O2 called TL – 1 for ca. ten minutes. After that it was time for the second step

where the surfaces were boiled for ten more minutes in a new mixture, called TL – 2, that solution contained 6 parts H2O, one part HCl and one part H2O2.

After those two steps the surfaces were very hydrophilic but to make it easier for the insulin protein to stick to the surfaces and make fibrils on them they had to be hydrophobic. This was done by first drying them with N2 gas and then putting them for five minutes in so called DDS

solution. It is a mixture of 1% DDS or dichlorodimetylsilane in xylene.

Finally the surfaces were ultrasonicated in xylene for ten minutes to shake of the possible impurities that could be left.

Now when the surfaces were clean and hydrophobic it was time for the incubation step where insulin fibrils are sticking to the surfaces making a thin layer that later on will be used as a catalyst to speed up the fibrillation process in the kinetic experiment.

To have a reference to the thickness of the surfaces I started with measuring them with an ellipsometer before the incubation step.

The ellipsometer measures the thickness with a laser beam that is reflected off the surface. Through the difference of polarization of the light reflected, that is then captured by a detector it gives the thickness of the surface4. Since silicon is a compound where the light can reflect and the laser beam do not go through it, it is a very effective way of measuring the thickness but when using the ellipsometer on glass the laser beam reflects in all kinds of different directions and even go through the glass making it impossible for the detector to capture all fractionated light beams and thereby making it difficult to get a correct view of the thickness of glass. However since the experiment with the glass was completely new I decided to try it without knowing how thick the surface was from the beginning and how thick the layer were going to be later on, just to see if it does affect the fibrillation kinetics. Although in this case with the glass surface I will not be able to prove that the thickness of the fibril layer affect the kinetics as with the silicon surfaces were it has been proved before me, that a thicker layer leads to faster fibrillation.

I relayed on the previous experiment done with hydrophobic silicon surfaces, but also my own eyes because the fibrils growing on the surface could be seen with the bare eye. The starting thickness of the two silicon surfaces were 17 respectively 18,6 Å.

With the reference thickness ready each surface was placed in an eppendorf tube and covered with fresh insulin solution with a concentration of 200 µM.

Then the tubes were incubated in a heating block at ca. 60˚C. At certain times I carefully took out the surfaces with a tweezers, dried them with N2 gas and measured the thickness. The step

where the surfaces are taken up in the air and dried with N2 gas is important since it has been

proved to be necessary to speed up the growth of insulin fibrils on the surfaces. It is also important to continuously add fresh solution so that there are enough free insulin monomers to stick to the surface.

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It has been shown previously that when growth on the surfaces reaches a crucial point where there are no more monomers left in the solution a sudden decrease of the fibril layer starts and ends when the starting layer, the one that is built up after the first incubation time is reached.4 Because of this it is important to constantly measure the thickness and to not leave the surfaces for a too long time in the solution since you will end up where you started with no difference in growth.

The thickness I was going for was around 200 Å. I also wanted one surface to be thicker than the other to prove what have been said before, that the thicker the surface the faster the fibrillation of insulin goes. When I was done with the incubation I had one 180 Å thick surface and one 500 Å thick surface. I also had a glass surface of unreliable thickness. A table of the incubation measurements is seen below (Table 1). Thickness measurements of the glass surface are also seen just to prove that the measurements are not consistent.

Time (min) Thickness of silicon surface 1 (Å) Thickness of silicon surface 2 (Å) Thickness of glass surface (Å) 0 17 19 1408 15 21 30 1408 120 21 30 1408 135 21 30 1408 160 60 44 1440 170 180 200 1406 190 only silicon 2 500 (Table 1) 4.1.3. Kinetic experiment

The first step of the incubation of insulin solutions for the kinetic experiment started with making a time pattern for the heating of blank solution in the heating block with the volumes I was going to use. (See table 2 below)

Temperature (˚C) Heating time for 1000 µ (min)

Heating time for 500 µ (min) 57 4,8 3,4 60 5,7 4,2 62 8 5

(Table 2) Then I prepared six Nunc tubes with different solutions and different volumes. The tubes contained:

A – 500 µl pure 200 µM stock insulin B – 1000 µl pure 200 µM stock insulin

C – 1000 µl 200 µM stock insulin with 200 Å layer on a silicon surface D – 1000 µl 200 µM stock insulin with 500 Å layer on a silicon surface E – 1000 µl 200 µM stock insulin with the glass surface

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4.1.4. Fluorescence measurements

The following step I was going to do was very time dependant. If it was possible it would have been desirable to take all the samples at the same time. However it is impossible but the samples were taken with the minimum time difference. During the whole experiment the heat block temperature was held constant.

I first took the samples at the starting point, meaning every tube was vortexed and samples of 4 µl of solution were taken from every tube, before incubation at 60˚C. The samples were pipetted to a microtiter plate.

It is very important to change the tip of the pipette between every sample since every tube contains different amount of fibrils that are formed in the tube. Besides that, it is also important to vortex every tube before the samples are taken to resuspend formed fibrils. When the first samples were taken I putted the Nunc tubes in the heating block and at certain times took out one tube at time vortexed it, and took another 4 µl to a well at the microtiter plate.

In the beginning of incubation the samples were taken very often, but later on when I took the samples every hour, the tubes that were incubated was covered with aluminium foil and the microtiter plate was stored in the refrigerator covered with parafilm to prevent evaporation. When all the samples of the incubated insulin probes were taken to the wells of the microtiter plate, 200 µl of 4 µM ThT solution was added making it able to measure the fluorescence intensity of the protein as mentioned before.

The tables below (tables 3-9) show the times the samples were taken and the fluorescence intensity at 490 nm. Since the samples were taken over a long period of time 2 microtiter plates had to be used. The samples named AA, BB, CC, DD, EE, and FF come from the second plate and are just an extension of the first plate. Where AA is 500 µl pure 200 µM stock insulin, BB is 1000 µl pure 200 µM stock insulin, CC is 1000 µl 200 µM stock insulin with 200 Å silicon surface, DD is 1000 µl 200 µM stock insulin with 500 Å silicon surface, EE is 1000 µl 200 µM stock insulin with the glass surface and FF is 1000 µl 200 µM stock insulin with 0,17% seeds.

Also six reference samples were taken in the last wells containing only ThT.

The values of fluorescence intensity, were plotted against the time giving a good view of how the kinetics of insulin fibrillation is affected by the different additives I had. (See graph 1 and 2 below)

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(Table 3) (Table 4) (Table 5) (Table 6) Well Fluorescence intensity at 490 nm Time (min) A1 158 0 A2 121 2 A3 107 5 A4 137 8,07 A5 165 12 A6 113 20 A7 168 30 A8 156 45 A9 170 60 A10 227 120 A11 444 180 A12 1240 240 AA1 6309 300 AA2 54533 360 AA3 46556 420 AA4 65535 480 Well Fluorescence intensity at 490 nm Time (min) B1 124 0 B2 108 2,6 B3 107 5,42 B4 97 8,5 B5 128 12,42 B6 127 20,43 B7 137 30,48 B8 171 45,4 B9 137 60,38 B10 173 120,47 B11 130 180,45 B12 151 240,52 BB1 166 300,7 BB2 252 360,45 BB3 463 420,52 BB4 1576 480,47 Well Fluorescence intensity at 490 nm Time (min) C1 164 0 C2 139 3,07 C3 164 5,88 C4 419 8,9 C5 369 12,82 C6 2771 20,91 C7 1039 30,95 C8 1368 46,07 C9 8753 61 C10 5898 121 C11 5127 181 C12 9865 241,75 CC1 6576 301,25 CC2 93 361 CC3 7936 420,9 CC4 7382 481 Well Fluorescence intensity at 490 nm Time (min) D1 135 0 D2 251 3,55 D3 2096 6,27 D4 748 9,33 D5 1536 13,17 D6 1314 21,32 D7 1778 31,35 D8 2017 46,67 D9 31824 61,5 D10 24740 121,53 D11 7437 181,42 D12 12203 241,75 DD1 58172 302 DD2 16317 361,47 DD3 13092 421,35 DD4 23954 481,28

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(Table 7) (Table 8) (Table 9) Well Fluorescence intensity at 490 nm Time (min) E1 131 0 E2 127 3,95 E3 122 6,73 E4 128 9,73 E5 133 13,52 E6 153 21,77 E7 148 31,83 E8 163 46,97 E9 224 62 E10 438 122 E11 1984 181,93 E12 7931 242,58 EE1 10500 302,58 EE2 7724 362,08 EE3 5460 421,93 EE4 10611 481,67 Well Fluorescence intensity at 490 nm Time (min) F1 132 0 F2 126 4,37 F3 148 7,15 F4 140 10,15 F5 148 13,93 F6 163 22,23 F7 201 32,17 F8 267 47,43 F9 431 62,67 F10 988 122,65 F11 8205 182,58 F12 8223 243 FF1 8908 303,12 FF2 13343 362,7 FF3 9085 422,45 FF4 16045 482,08 Well Fluorescence intensity at 490 nm Time (min) AA5 (ref.) 65 0 BB5 (ref.) 71 0 CC5 (ref.) 64 0 DD5 (ref.) 85 0 EE5 (ref.) 68 0 FF5 (ref.) 65 0

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Insulin fibrilatin (200uM) 000 00 100 00 200 00 300 00 400 00 500 00 600 00 700 00 0 100 200 300 400 500 time (min) fl u o resce n ce i n ten si ty at 490 n m A(500ul) B(1000ul) C(1000ul,200Å) D(1000ul,500Å) E(1000ul,glass) F(1000ul,seeds) (Graph 1)

Insulin fibrilatin (200uM)

000 00 010 00 020 00 030 00 040 00 050 00 060 00 070 00 0 100 200 300 400 500 time (min) fl u o resce n ce i n ten si ty at 490 n m A(500ul) B(1000ul) C(1000ul,200Å) D(1000ul,500Å) E(1000ul,glass) F(1000ul,seeds) (Graph 2)

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As seen from the graphs this first experiment did not go very well. But still the main purpose of the experiment was to monitor rate of fibrillation. It can be seen if we look at the beginning of the graph that the lag-phase is shortened and the hydrophobic surfaces are most effective for enhancing the fibrillation of insulin (see graph 2). The thicker coated surface is more effective then the thinner but the glass surface is most effective probably because it had a coating thicker than both silicon surfaces. What also can be seen is that all the surfaces are more effective than regular amyloid seeds and that the seeds in comparison with plain insulin solution are shortening the lag-phase.

After the kinetic experiment the used surfaces were first washed with 25 mM HCl and distil H2O and then stained with the amyloid specific dye Congo red, making it possible to see the

insulin fibril depositions in an optical microscope. Beside the two silicon surfaces that were used in the kinetic experiment, one “empty” surface, a surface that was not pre-coated but incubated for 24 hours with insulin under amyloid conditions, (in 25 mM HCl, 500 µM insulin at 65˚) was stained to see the difference between the empty surface and the two other where insulin was attached to it. The pictures of the surfaces can be seen in the appendix I – III on pages 27 - 34.

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4.2. Experiment 2

4.2.1 Making the stock solution

Since the first experiment was a bit to noisy, I had to do another set of the same experiment. For this experiment I did not have the same amount of insulin to start with as before. So I used what was left in the insulin jar and dissolved it. However in the end it did not differ that much from the first experiment so the same amount of GuHCl was used to dissolve. In the same way the solution was put on dialysis against cold 25 mM HCl under constant stirring for at least eight hours in 4˚C and finally filtered through a 0,45 µm filter to a Nunc tube. Also this time I measured UV spectra between 250 and 400 nm and the peak at 277 nm was used to calculate the concentration. Since I suspected that the concentration of insulin was lower this time I diluted the solution for the spectrophotometric measurements only three times. ABS_ x times diluted = concentration in M

Ex.coeff

2,505 x 3 = 0,0013 M = 1,3 mM 5747

Since this peak of absorbance is near ABS = 3.0 it could give incorrect values so I diluted the stock solution three fold more and then out of that I took enough of insulin to diluted it three more times, totally nine for the absorbance measurement again. This time I got

0,423 x 9 = 0,00067 M = 0,67 mM 5747

From this concentration value I diluted enough insulin to a concentration of 200 µM, up to a volume of 7.5 ml. 2 ml were going to be used for the preparation of the surfaces this time because I was going to make 2 glass and three silicon surfaces, and the rest for the incubation part of the kinetic experiment. The rest of the non-diluted insulin was stored in the

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4.2.2. Making the surfaces

The next step in the experiment is as before to make the hydrophobic surfaces. This time I needed three silicon surfaces with approximately the same thickness around 200 Å and two glass surfaces.

As before I started with cutting the surfaces approximately 5x5 mm with a diamond knife and then I cleansed them by the same procedure as before by boiling them first in TL – 1 and then in TL – 2, to then make them hydrophobic with N2 gas and 1% DDS. Finally they were

ultrasonicated in xylene to shake of the impurities

Like before it was then time for the incubation and I started with measuring the thickness of the surfaces. But this time I decided not to measure the glass surfaces.

The starting thickness of all three silicon surfaces was approximately 30 Å.

All five surfaces was then placed in eppendorf tubes and covered with fresh insulin solution with a concentration of 200 µM and incubated at approximately 60˚C.

At certain times I dried the surfaces with N2 gas and measured the thickness and continuously

added small amounts, approximately 130 µl of fresh monomers to the tubes.

Finally I had two glass surfaces and three silicon surfaces that were 230,240 and 270 Å thick. A table of the incubation measurements for the silicon surfaces is seen below (Table 10)

Time (min) Thickness of silicon

surface 1 (Å) Thickness of silicon surface 2(Å) Thickness of silicon surface 3(Å) 0 30 30 30 15 38 35 29 120 44 27 30 180 80 33 38 195 230 70 65 210 100 200 240 270 240 (Table 10)

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4.2.3. Kinetic experiment

Since the volumes I was using this time were the same as before 500 µl and 1000 µl

respectively and the heating block was the same, I did not have to do a new time pattern for the heating. The one I have already done was enough. (See table 2)

I then prepared seven Nunc tubes with different solutions and different volumes. The tubes contained:

A – 500 µl pure 200 µM stock insulin B – 1000 µl pure 200 µM stock insulin

C – 500 µl 200 µM stock insulin with 270 Å layer on a silicon surface D – 1000 µl 200 µM stock insulin with 240 Å layer on a silicon surface E – 500 µl 200 µM stock insulin with 0,17% seeds

F – 1000 µl 200 µM stock insulin with 0,17% seeds G – 1000 µl 200 µM stock insulin with glass surface And the kinetics experiment could begin.

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4.2.4. Fluorescence measurements

This following step is the time depending one where the samples as before have to be taken with the minimum time difference. The temperature was still held at 60˚C.

The first samples were taken at time zero, meaning every tube was vortexed and samples of 4 µl of solution were taken from every tube, before incubation at 60˚C to a microtiter plate. When the first samples were taken the Nunc tubes were placed in the heating block and at certain times I took out one tube at time vortexed it, and took another 4 µl to the microtiter plate.

As before when the waiting time was around one hour the tubes that were incubated was covered with aluminium foil and the microtiter plate was stored in the refrigerator covered with parafilm to prevent evaporation.

When all the samples of the incubated insulin solution were taken to the wells of the

microtiter plate, 200 µl of 4 µM ThT solution was added as before making it able to measure the fluorescence intensity of the protein.

When I was at my last row of sample wells unfortunately my ThT solution was finished. This created a pretty large problem to me since every ThT solution is slightly different because in ThT solution micelles are building up and depending on how old the solution is different amount is built up. What I then was going to do was to slowly take up the ThT solution from the first row of samples, (A), from the first microtiter plate that should not contain any fibrils and use them after the first microtiter plate was measured.

However I managed to make another mistake and take the ThT solution from the second plate that lead to the only solution I had to eliminate the first set of probes with 500 µl pure 200 µM insulin solution.

The tables below (Table 11-16) show the times the samples were taken and the fluorescence intensity at 490 nm. The values of fluorescence intensity, were plotted against the time giving a good view of how the kinetics of insulin fibrillation is affected by the different additives I had. (See graph 3 and 4 below) This time the graphs looked fine and the effects I was hoping to see could be seen but I noticed that my sample with pure insulin solution showed an unusually long lag-phase. Pretty soon I realized that the concentration of my samples seamed to be lower than it should be. Somewhere I had done a calculation mistake. I took what was left of the solution that I diluted and measured absorbance a couple of times. Every time I got a new value that was not even close to the one I got the first time. The spectrophotometer seamed to be playing me a trick. So to get more accurate concentration this time I took some of the solution I had left and measured it with the so called nano drop where only a few µl is enough to get a very precise value. It turned out that the concentration I used both for the kinetics and coating of the surfaces was only 64 µM. So the results below are done with an insulin solution with concentration of 64 µM. I also measured the concentration of the non-diluted solution I had left in the refrigerator and it turned out that it was more or less exactly 200 µM.

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(Table 11) (Table 12) (Table 13) (Table 14) Well Fluorescence intensity at 490 nm Time (min) B1 245 0 B2 29 2,25 B3 24 5,35 B4 25 8,38 B5 27 12,5 B6 24 20,37 B7 25 30,5 B8 29 45,25 B9 28 60,33 B10 24 120,33 B11 24 180,42 B12 31 240,42 B13 28 300,42 B14 29 360,25 B15 44 420,4 B16 78 480,33 Well Fluorescence intensity at 490 nm Time (min) C1 26 0 C2 42 2,58 C3 44 5,68 C4 61 8,7 C5 85 12,83 C6 92 20,67 C7 219 30,83 C8 440 45,58 C9 390 60,67 C10 1461 120,67 C11 3375 180,75 C12 2425 240,75 C13 2373 300,75 C14 2538 360,67 C15 1703 420,75 C16 2912 480,67 Well Fluorescence intensity at 490 nm Time (min) D1 36 0 D2 33 2,92 D3 30 6,17 D4 28 9 D5 36 13,12 D6 49 21 D7 102 31 D8 134 45,92 D9 169 61 D10 715 121 D11 1232 181,17 D12 1532 241,08 D13 3056 301,08 D14 2585 361 D15 2603 421 D16 2673 481 Well Fluorescence intensity at 490 nm Time (min) E1 307 0 E2 28 3,28 E3 31 6,5 E4 32 9,33 E5 35 13,42 E6 47 21,28 E7 50 31,5 E8 47 46,5 E9 57 61,5 E10 141 121,5 E11 233 181,58 E12 602 241,5 E13 2084 301,5 E14 2331 361,33 E15 2422 421,5 E16 2515 481,5

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(Table 15) (Table 16) Well Fluorescence intensity at 490 nm Time (min) F1 40 0 F2 52 3,75 F3 37 6,77 F4 41 9,67 F5 96 13,67 F6 82 21,58 F7 128 31,83 F8 141 46,83 F9 142 61,83 F10 327 121,67 F11 878 181,92 F12 1489 241,83 F13 2121 301,83 F14 2747 361,67 F15 1815 422 F16 2691 481,83 Well Fluorescence intensity at 490 nm Time (min) G1 1034 0 G2 110 4 G3 196 7 G4 269 10 G5 442 14 G6 727 21,83 G7 1250 32 G8 2283 47 G9 2721 62 G10 2798 122 G11 2597 182,25 G12 3427 242,2 G13 4770 302,08 G14 3107 362 G15 2637 422,33 G16 2431 482,17

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Insulin fibrillation (64uM) 0 1000 2000 3000 4000 5000 6000 0 100 200 300 400 500 600 time(min) fl u o rescen ce i n ten si ty at 490n m B(1000ul) C(500ul,270Å) D(1000ul,240Å) E(500ul,seeds) F(1000ul,seeds) G(1000ul,glas) (Graph 3)

Insulin fibrillation (64uM)

0 500 1000 1500 0 100 200 300 400 500 600 time(min) fl u o rescen ce i n ten si ty at 490n m B(1000ul) C(500ul,270Å) D(1000ul,240Å) E(500ul,seeds) F(1000ul,seeds) G(1000ul,glas) (Graph 4)

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Out of this graph it is possible to see that the amyloid coated glass is the most effective additive I used for fibrillation of insulin. When using the glass, the lag time, meaning the period of time before the fibrillation starts, seen from the graph the time period before the curve goes straight up, more or less totally disappear. From this it can be concluded that when adding a hydrophobic glass surface coated with amyloid-like fibrils to a solution of insulin the fibrillation starts right away probably because the glass surface have a thicker coating layer than the silicon surfaces. The values of the fluorescence intensity in the beginning of the fibrillation with the glass are pretty high to start with and then they fall down, this is most likely because of the thick coating layer of insulin on the glass where it speeds up the fibrillation at first but then as mentioned before reaches a critical thickness and buds off, followed by readsorption and another round of rebuilding a layer.

What also can be seen by comparing the results with different concentrations is that the low concentration of insulin does not seam to slow down the fibrillation when the surfaces are present. In the case with seeds it takes a little longer time to start the fibrillation but the difference is not that noticeable. As expected the thicker surface has a larger effect on the fibrillation rate and both surfaces have more effect than normal amyloid seeds. The large sample volume with two fold more seed particles is more effective than the smaller and pure insulin fibrillate in longest time.

The 240 Å surface that was used for the kinetic incubation was as before washed in 25 mM HCl and distilled H2O and stained with Congo red as was the 230Å surface making it possible

to compare one only insulin coated and one coated surface used for the kinetic incubation. Also one coated glass surface and the glass surface from the kinetics were stained. The pictures can be seen in appendix IV-V on page 35-38.

To in some way fix the mistake I did with using lower concentration of insulin and for taking out ThT solution from wrong wells, I did the kinetic experiment for three more samples. One diluted three times to the solution I used previously to compare the different concentrations and two without dilution to see that the surface, in this case a reused one, would still be more effective than plain insulin.

A – 500 µl pure 200 µM stock insulin

B – 500 µl 200 µM stock insulin with already once used 270 Å silicon surface C – 500 µl pure 64 µM stock insulin

The same procedure was done once again were I started with taking samples at the starting point at time zero to then follow the same pattern of taking out the Nunc tubes, vortex them and take out 4 µl samples to a microtiter plate. This time I did not make a fresh silicon surface. Instead I washed the 270 Å thick surface, first in 25 mM HCl and then in distil H2O

and reused it for the kinetic incubation.

The following tables (Tables 17-20) and graphs (Graph 5 and 6) are the result of the incubation. The well B8 is clearly giving me a misleading result since it differs that much from the rest, so in the graphs it has been removed. Also the end time in this experiment is longer than before because I wanted to see if the insulin in the low concentration sample will start to fibrillate.

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500 µl, 200 µM 500 µl, 200 µM, recycled 270 Å silicon surface (Table 17) (Table 18) 500 µl, 64 µM Blank (Table 20) (Table 19) Well Fluorescence intensity at 490 nm Time (min) B1 1900 0 B2 2383 2,28 B3 4321 5,37 B4 6970 8,28 B5 9121 12,3 B6 11395 20,35 B7 8472 30,28 B8 53087 45,32 B9 9071 60,32 B10 11177 120,38 B11 7338 180,27 B12 12191 240,37 B13 8640 300,32 B14 9669 360,37 B15 4451 420,27 B16 6350 480,33 B17 9555 1620,3 Well Fluorescence intensity at 490 nm Time (min) A1 22 0 A2 22 2 A3 22 5 A4 23 8 A5 20 12 A6 24 20 A7 25 30 A8 34 45 A9 33 60 A10 31 120 A11 90 180 A12 791 240 A13 1100 300 A14 1377 360 A15 2537 420 A16 2012 480 A17 2143 1620 Well Fluorescence intensity at 490 nm Time (min) C1 35 0 C2 34 2,6 C3 34 5,67 C4 34 8,65 C5 32 12,68 C6 38 20,7 C7 34 30,42 C8 30 45,67 C9 38 60,67 C10 34 120,4 C11 42 180,6 C12 41 240,77 C13 45 300,6 C14 56 360,75 C15 124 420,62 C16 234 480,73 C17 824 1620,7 Well Fluorescence intensity at 490 nm Time (min) A18 31 0 B18 40 0 C18 37 0

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Insulin fibrillation 0 2000 4000 6000 8000 10000 12000 14000 0 500 1000 1500 2000 time(min) fl u o rescen ce i n ten si ty at 490n m A(500ul,200uM) B(500ul,200uM,270Å recycled) C(500ul,64uM) (Graph 5) Insulin fibrillation 0 1000 2000 3000 4000 5000 6000 0 500 1000 1500 2000 time(min) fl u o rescen ce i n ten si ty at 490n m A(500ul,200uM) B(500ul,200uM,270Å recycled) C(500ul,64uM) (Graph 6)

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Even though this graph is not perfect since the recycled insulin coated silicon surface is as efficient as the coated glass surface earlier, where it starts at very high intensity values to then fall down, because of the same reason that the fibril surface is very thick in the beginning, then reduced when it reaches the critical thickness and finally gradually built up again. It can bee seen as before that the fibrillation of the protein is much more effective with a surface present and with higher concentration of insulin giving a shorter lag-phase.

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5. Difference between washed and not washed stained surface

Except these kinetic experiments I also wanted to see what effect the washing of the surfaces before staining them in Congo red had.

To this experiment I simply took a 70 Å insulin coated silicon surface directly from the insulin solution to the Congo red dye, left it to be stained and later on I dried the surface with N2 gas and looked at the surfaces with an optical microscope. Large depositions could be

seen. (See pictures in appendix VI on page 39-40)

When the first staining was done and the surfaces were investigated I took the same surface and washed it with 25 mM HCl and distil H2O, dried it with N2 gas and looked at it once

again with the microscope. This time almost all of the insulin depositions was gone leaving a thin layer of birefringent particles. (See pictures in appendix VII on page 41-42)

To make sure that I washed away the non sticky insulin fibrils and not simply washed away the Congo red dye I once again placed the surface in the Congo red and left it to be stained. The third time I looked at the surface slightly more insulin depositions could be seen than the time before but not as much as before washing the surface.

From these experiments I could conclude that if the surfaces are not washed before they are stained they could give a misleading picture of the actual insulin depositions that are attached to the surface. (See pictures in appendix VIII on page 43-44)

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6. Discussion and source of error – what could have been done better?

The whole purpose of the experiment was to see if the conclusions drawn before, that insulin coated hydrophobic surfaces enhance the amyloid fibril formation of insulin, were true. As can be seen from the results of both sets of experiments it is a correct conclusion.

In contact with hydrophobic surfaces such as coated silicon or glass surfaces insulin protein is fibrillating much faster than normally under conditions where amyloid fibrillation can

proceed, such as a temperature of 60˚C and pH 1,6.

Why glass surfaces are much more effective is probably because when they are coated they gain a much thicker fibril layer than silicon. The reason why the fibril coating is larger on glass than on silicon even though the incubation time is the same may be that the glass in contrast to silicon has a less even surface that automatically means that the surface area is larger. If we look at the pictures of silicon surfaces they have two sides. One smooth side and one so called grey side. The grey side is as the glass surface less even and should give more hydrophobic area. As seen from the pictures of the grey side more protein is sticking, which I also see as an indication to the fact that a surface of same size but more uneven gives a larger area for the coating and thereby a larger hydrophobic area for the protein to stick to, leading to increased fibrillation during the incubation. Another indication that the fibril layer is thicker on glass is what can be seen in graph 3 and 4 where the glass curve starts at a high intensity value meaning that the fibrillation starts immediately. After the initial “burst” the fibrillation dramatically decreases where probably the critical thickness of the layer has been reached and the fibrils fall back to the solution and then it slowly increases again. This behaviour can also be seen with the recycled silicon surface that when used for the second incubation already have a thick layer.

Normal preformed amyloid seeds are also efficient even if not as effective as the surfaces in such low concentrations that I used.

The mistake that I made when calculating the concentration wrongly in the second

experiment, in a way became a positive mistake since I could see how effective the coated hydrophobic surfaces really are. Even though I hade a three time lower concentration of

insulin both for the coating and for the kinetic incubation the result was more or less the same. With a concentration of insulin that would need a couple of days to normally fibrillate in 60˚C and pH 1,6 in presence of hydrophobic silicon or glass surfaces this process takes less then four hours.

Since the glass surface is much easier to analyze under microscope I am glad that it worked, that the effect on the fibrillation was the same. A problem with the glass surfaces is that the thickness was not possible to measure so the coating incubation time could not be adjusted to glass. Maybe this process takes less time than with the silicon surfaces. Another problem is since the glass gained such a thick layer it was a little bit tricky to look at it under the microscope since there were many deposited layers that covered each other.

When staining the surfaces it is important to wash them first in 25 mM HCl and distil H2O

before placing them in Congo red dye. Otherwise the depositions seen with the microscope will be misleading since all the insulin will not be attached to the hydrophobic surface but simply loosely adsorbed to it.

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7. References

1. Atta Ahmad, Vladimir N. Uversky, Dongpyo Hong, Anthony L. Fink.

Early Events in the Fibrillation of Monomeric Insulin. The journal of biological chemistry, v 280, no.52, December 2005, pp 42669-42675,ISSN 00219258

2. Liza Nielsen, Ritu Khurana, Alisa Coats, Sven Frokjaer, Jens Brange, Sandip Vyas,

Vladimir N. Uversky, Anthony L. Fink. Effect of Environmental Factors on the Kinetics of Insulin Fibril Formation: Elucidation of the Molecular Mechanism. Biochemistry, v 40, no.20, May 2001, p6036-6046.

3. Umesh Masharani, MB, BS, MRCP(UK), Michael S. German, MD. Lange Endocrinology,

chapter 18, Pancreatic Hormones & Diabetes Mellitus, Hormones of the endocrine pancreas, Insulin.

URL:http://www.accessmedicine.com.lt.ltag.bibl.liu.se/content.aspx?aID=2633161&searchSt r=function+of+insulin#searchTerm

(2007 – 06 – 11)

4. Per Hammarström, Malik M. Ali, Rajesh Mishra, Pentti Tengvall, Samuel Svensson,

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Appendix I

Silicon surfaces seen by a magnifying glass from the first set of experiments – all pictures are

enhanced

(grey side = non shining side, empty surface = non-coated surface, incubated for 24 h at amyloid conditions of 25mM HCl, 500µM insulin at 65˚)

Silicon surface 500Å grey side, 100x Silicon surface 500Å, 100x

Silicon surface 500Å grey side, 100x Silicon surface 500Å, 46.5x

Silicon surface 180Å, 100x Silicon surface 180Å, 100x

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Silicon surface 180Å grey side, 100x Empty silicon surface grey side, 100x

Empty silicon surface, 100x Empty silicon surface, 100x

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Appendix II

Silicon surfaces seen by a microscope from the first set f experiments – all pictures are

enhanced

(grey side = non shining side, empty surface = non-coated surface, incubated for 24 h at amyloid conditions of 25mM HCl, 500µM insulin at 65˚)

Silicon surface 500Å, 20x Silicon surface 500Å grey side, 4x

Silicon surface 500Å grey side, 20x Silicon surface 180Å, 4x

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Silicon surface 180Å grey side, 20x Silicon surface 180Å grey side, 20x

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Empty silicon surface, 20x Empty silicon surface, 20x

Empty silicon surface, 20x Empty silicon surface grey side, 20x

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Empty silicon surface grey side, 20x Empty silicon surface grey side, 20x

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Appendix III

Glass surface seen by a microscope from the first set of experiments – all pictures are

enhanced

Glass surface, polarization 1, 4x Glass surface, polarization 2, 4x

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Glass surface, polarization 1, 20x Glass surface, polarization 2, 20x

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Appendix IV

Silicon surfaces seen by a microscope from the second set of experiments – all pictures are

enhanced

(grey side = non shining side, ref. surface = coated surface, not used for the kinetics 230Å) Ref. silicon surface, 20x Ref. silicon surface, 20x

Ref. silicon surface grey side, 20x Silicon surface 240Å, 4x

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Appendix V

Glass surface seen by a microscope from the second set of experiments – all pictures are

enhanced

(ref. surface = coated surface that has not been used for the kinetics)

Glass surface polarization 1, 20x Glass surface polarization 1, 20x

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Ref.glass surface polarization 1, 20x Ref.glass surface polarization 1, 20x

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Appendix VI

Silicon surfaces seen by a microscope from the staining experiments, non-washed surfaces – all pictures are enhanced

(grey side = non shining side)

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Appendix VII

Silicon surfaces seen by a microscope from the staining experiments, washed surfaces – all pictures are enhanced

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Appendix VIII

Silicon surfaces seen by a microscope from the staining experiments, second round of staining – all pictures are enhanced

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