Examples of Phase DiagramsExamples of Phase Diagrams

0 10 20 30 40 50 60 70 80 90

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 10 20 30 40 50 60 70 80 90

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 10 20 30 40 50 60 70 80 90

338

339

340

341

342

343

344

345

346

20 30 40 50 60 70

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

0 10 20 30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70 80 90

496

498

500

502

504

506

508

510

512

514

516

518

520

522

524

0 10 20 30 40 50 60 70 80 90

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70 80 90 100

6

8

10

12

14

16

A

Re

lativ

e M

E a

t 2

22

nm

Temperature (oC)

B

Re

lativ

e F

l. p

ea

k in

ten

sity

Temperature (oC)

C

Fl.

pe

ak

po

sitio

n (

nm

)

Temperature (oC)

D

Re

lativ

e h

elix

co

nte

nt

Temperature (oC)

E

Re

lativ

e A

NS

Fl.

pe

ak

inte

nsi

ty

Temperature (oC)

F

AN

S F

l. p

ea

k p

osi

tion

(n

m)

Temperature (oC)

G

Op

tica

l de

nsi

ty a

t 3

50

nm

Temperature (oC)

H

Re

lativ

e F

l. lig

ht

sca

tte

rin

g

Temperature (oC)

Figure 1

I

Exc

ess

MH

C

Temperature (oC)

0 10 20 30 40 50 60 70 80 90

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 10 20 30 40 50 60 70 80 90

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 10 20 30 40 50 60 70 80 90

339

340

341

342

343

344

345

346

347

348

349

350

351

20 30 40 50 60 701.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

0 10 20 30 40 50 60 70 80 900.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70 80 90

500

502

504

506

508

510

512

514

516

518

520

522

524

0 10 20 30 40 50 60 70 80 900.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70 80 90

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70 80 90 100

0

2

4

6

8

10

12

14

16

18

20

A

Re

lativ

e M

E a

t 2

22

nm

Temperature (oC)

B

Re

lativ

e F

l. p

ea

k in

ten

sity

Temperature (oC)

C

Fl.

pe

ak

po

sitio

n (

nm

)

Temperature (oC)

D

Re

lativ

e h

elix

co

nte

nt

Temperature (oC)

E

Re

lativ

e A

NS

Fl.

pe

ak

inte

nsi

ty

Temperature (oC)

F

AN

S F

l. p

ea

k p

osi

tion

(n

m)

Temperature (oC)

G

Op

tica

l de

nsi

ty a

t 3

50

nm

Temperature (oC)

H

Re

lativ

e F

l. lig

ht

sca

tte

rin

g

Temperature (oC)

Figure 2

I

Exc

ess

MH

C

Temperature (oC)

Interpretation

Experimental data Experimental data visualization visualization

VisualizationVisualization

Reconstructed 3-D Vector 321, xxxv i TpH

Rescale the vector to RGB color system

10 '''' '321, i

iTpH xxxxv

Each vector will define a color block in the phase diagram at its corresponding coordinates (pH, T)

'

'

'

3

2

1

x

x

x

B

G

R

Phase diagram created using high-resolution Phase diagram created using high-resolution second derivative UV spectroscopy data. second derivative UV spectroscopy data.

4 5 6 7 8pH

10

20

30

40

50

T Ricin2nd DerivativeUV

Phase diagram generated using ANS fluorescence and Phase diagram generated using ANS fluorescence and intrinsic Trp fluorescence intensity data and CD molar intrinsic Trp fluorescence intensity data and CD molar

ellipticity data of recombinant ricin toxin A-chain. ellipticity data of recombinant ricin toxin A-chain.

4 5 6 7 8 9pH

20

40

60

80

T Ricin ANS Int., Intrinsic Int., & CD

Phase diagram rPA based on intrinsic, ANS Phase diagram rPA based on intrinsic, ANS binding fluorescence and CDbinding fluorescence and CD

3 4 5 6 7 8 9pH

20

40

60

80

T Antrax PA # 1

2

Stable phase

Structrally altered and aggregated state

Molten globule state

Severely structurally altered phase

Empirical phase diagram (EPD) of Empirical phase diagram (EPD) of RSV over the pH range 3-8RSV over the pH range 3-8

Data included in the generation of the EPD are all negative second-derivative UV peaks, CD signal at 222 nm, optical density at 350 nm, intrinsic fluorescence peak position, intrinsic fluorescence intensity at 330 nm, and ANS fluorescence intensity at 485 nm

Ausar, et al, Mol. Pharm., 2 (6), 491 -499, 2005.

Adenovirus Type 2Adenovirus Type 2

Non-Viral Gene Delivery Vehicle Non-Viral Gene Delivery Vehicle DOTAPDOTAP

Non-Viral Gene Delivery Vehicle DOTAP Non-Viral Gene Delivery Vehicle DOTAP (Charge ratio =4.0)(Charge ratio =4.0)

Non-Viral Gene Delivery Vehicle Non-Viral Gene Delivery Vehicle DOTAP/DOPEDOTAP/DOPE

Non-Viral Gene Delivery Vehicle Non-Viral Gene Delivery Vehicle DOTAP/DOPE (Charge ratio = 4.0)DOTAP/DOPE (Charge ratio = 4.0)

Non-Viral Gene Delivery Vehicle PLL Non-Viral Gene Delivery Vehicle PLL (Charge ratio =4.0)(Charge ratio =4.0)

Ty21a bacteriaTy21a bacteria

HSPPC96HSPPC96

HSPPC96 Visual InterpretationHSPPC96 Visual Interpretation

HSC70HSC70

HSC70 Visual InterpretationHSC70 Visual Interpretation

FGF1FGF1

FGF1, heparin excipientFGF1, heparin excipient

FGF1, sucrose excipientFGF1, sucrose excipient