Applications of Hydrostatic Pressure in Chemical and

Biomedical Analysis: Exploring

a Well Forgotten Thermodynamic Dimension

Alexander Lazarev, Ph.D.

ANALYTICA Biotech Forum April 18th, 2012

12:30 – 13:00

IUL Instruments B1-110; A3-110

Two Dimensions of Thermodynamics:

Ichimori H. et al., 1999; in: Advances in High Pressure Bioscience and Biotechnology,

Horst Ludwig (Ed.), Proceedings of the Intl. HPBB Conference, Heidelberg, 1998.

A) Pressure-induced phase transitions of water

B) Interdigitation of lipid bilayers in an ester-ester linked HPPC

bilayer: High Pressure DSC data.

Pressure cycling at 33 ºC

A B

Why High Pressure?

• Pressure is a fundamental parameter that governs

the structure of matter and chemical reactions.

Pressure is particularly important for the structure

and interactions of complex biological molecules.

• Pressure can be controlled with high accuracy, is

transmitted at sonic speeds, and can be applied

and removed at rapid rates. Unlike heat, there is

no thermal diffusion time.

Reaction Activation Volume and The Effect of Hydrophobic Hydration

“Thermodynamic state of solvent water surrounding

the exposed hydrophobic groups plays a

decisive role in determining the thermodynamic

stability of a protein.”

Yamaguchi et al., J. Mol. Biol. (1995) 250, 689-694

H2O → H+ + OH- Δ‡V = - 21.3 mL/mol

(CH4)hexane → (CH4)water ∙ H2O Δ‡V = - 22 mL/mol

Myoglobin denaturation Δ‡V = - 98 mL/mol

Lactate Dehydrogenase M4 → 4M Δ‡V = - 500 mL/mol

Percy Bridgman –

The Nobel Prize in Physics, 1946

• Professor at Harvard University

• Invented methods of working with extreme pressure

• Reported on albumin, hemoglobin and other

biological materials are affected by pressure

•Used mercury “piston” in

small glass tubes to

transmit pressure to sample

• Kerosene was used as the

hydrostatic pressure media

Temperature vs. pressure control equipment

High Pressure Applications

1. Pressurized discharge

2. Pressurized flow (HPLC)

3. HPP inactivation

4. Cell lysis and dissolution

5. Molecular perturbation

6. Enzyme control

7. Environmental stress

8. Adiabatic heating/cooling

9. Solubility change

10. Freezing suppression

Protein effects

Phase

change effects

Mechanical stress

Compression effect

“Cycles of hydrostatic pressure between ambient

and high levels, enabling precise control of

molecular interactions”

0

5000

10000

15000

20000

25000

30000

35000

3:56:10 3:56:53 3:57:36 3:58:19 3:59:02 3:59:46

Pressure Cycling Technology (PCT):

1.1 kbar

(Marianas

Trench)

Pressure, psi

Time, h:mm:ss

Pressure Biosciences Products

BarocyclerTM NEP3229 BarocyclerTM NEP2320

PULSE Tube FT500

PULSE Tube FT500-ND

PCT Shredder PCT MicroTubes

BarocyclerTM HUB440

Reagent

Kits

Sample Containment

Sample containers are either flexible or incorporate a displacement means

Moving piston

Flexible tubes

Barocycler: Sample Preparation Applications

www.pressurebiosciences.com

101 241 40 652 108 180

proteins peptides

Current install base – ca. 200 systems worldwide

High pressure inactivation

Inactivation has been studied widely in the food

industry. Pressure denaturates several key

proteins necessary to sustain reproduction and

metabolism of food-borne pathogens and

organisms causing product spoilage.

• Target dependent

• Media dependent

• Temperature dependent

• Synergistic with denaturants

Sample preparation for biological threat agents

Inactivation for molecular analysis

BSL3/4 to BSL2 analysis

Pressure destabilizes biological membranes

Hydrostatic Pressure

Applied Hydrostatic Pressure

Released*

Lip

id b

ilaye

r

Mem

bra

ne

Pro

tein

(Interdigitated bilayer,

Hydrophobic hydration)

Gross, et al., Journal of Biomolecular Techniques (2008) 19:187–197

*Appropriate solvent system is required for lysis

DNA extraction from soil pathogens

P. Okubara, USDA ARS, Pullman WA

Mitochondrial DNA from single hair

Gonzalez, Feller, Peters, Budowle, and Eisenberg

University of North Texas Health Science Center

(2 cm)

Differential cell lysis

Barocycler Highlighted in an opening

episode of CSI - New York, 2012

PCT-mediated liquid-liquid extraction (patent pending)

1 5 4 3 2

a

b

a

b

a

b

c c

a

b b

a

Pressure Applied

Hydrophobic hydration and intermixing of poorly miscible solvents under

pressure enables extraction in the homogenous solvent phase followed by

partitioning under atmospheric pressure conditions.

Gross, et al., Journal of Biomolecular Techniques (2008) 19:187–197

PCT-Dependent Detergent-

Free Extraction of Proteins,

DNA and RNA

Effect of high pressure on enzymatic activity

0

20

40

60

80

100

0 100 200 300 400

Pressure (MPa)

LDH AST

ALT

Amylase

Lipase

Alk P’ase

Enzymes under pressure

Pressure can selectively increase or reduce activity of certain

enzymes:

• Early efforts in DNA sequencing (Exonucleases)

• Inactivation of enzymatic activity during cell lysis and

extraction (Phosphatases, Proteases, RNases, etc.)

• Proteolytic digestion in proteomic sample preparation

(Trypsin, Chymotrypsin, Pepsin, Lys-C)

• Protein removal for cell lysis and DNA purification

(Lysozyme, Proteinase K, Zymolyase)

• Enzymatic protein deglycosylation (PNGase F)

Barocycler, 1h Thermomixer, 18 h

Unique Peptides 832 288

Unique Proteins 342 141

Integral Membrane Proteins 62 15

Digestion of Membrane Proteins at 1.4 kbar

101 241 40

Proteins

180 652 108

Peptides

12 50 3

Integral Membrane Proteins

Data: Eric Bonneil and Pierre Thibault, University of Montreal, 2010

0.00E+00

4.00E+05

8.00E+05

1.20E+06

1.60E+06

2.00E+06

MQIFVK TITLEVEPSDTIENVK LIFAGK TLSDYNIQK1:100 1:50 1:10 1:100 1:50 1:10 1:100 1:50 1:10 1:100 1:50 1:10 1:100 1:50 1:10 1:100 1:50 1:10 1:100 1:50 1:10 1:100 1:50 1:10

MQIFVK▲TLTGK▲TITLEVEPSDTIENVK▲AK▲IQDK▲EGIPPDQQR▲

LIFAGK▲QLDGR▲TLSDYNIQK▲ESTLHLVLR▲LR▲GG

Trypsin digestion of ubiquitin: PCT at 1.4 kbar vs Atmospheric Pressure Control

Deglycosylation of IgG with PNGase F enzyme:substrate ratio - 1:2500

Oligosaccharide Ladder

IgG glycans, 2 kbar. 5 min.

IgG glycans, 1 Atm. 3 hours.

IgG glycans, 1 Atm. 5 min.

Szabo, Z., et al., Analytical Chemistry, 82 (6), pp. 2588-2593, 2010

PCT workflow: mitochondrial extraction and enrichment

Fresh Tissue Sample

Tissue-

specific

kits

NIGMS SBIR-II GM079059

Gross VS et al., Analytical Biochem. 418(2):213-23, 2011

Pressure cycling

at 0.6-1kbar

High pressure molecular perturbation

• Allows to directly, and reversibly modify Gibbs free energy of the

system without addition of thermal or chemical energy to investigate

fundamental aspects of macromolecular structure dynamics

• Stabilizes high-energy intermediates

• Perturbs ligand binding

• Frequently combined with analytical (optical and electromagnetic)

spectroscopic methods

High Pressure

• Unique Plug-and-Play instrument

• USB-powered computer interface

• Sophisticated pressure control

• Data logging

• External triggering

A broad selection of custom

options is available from

Pressure BioSciences,

your trusted supplier of high

pressure equipment and

consumable products.

Applications – high pressure

perturbation for magnetic resonance

and optical spectroscopy, chemical

synthesis and material science.

Barocycler HUB440 –

plug-and play bench-top high pressure research

Modular design for multiple applications

• Programmable bench-top high

pressure generator and

accessories (300 – 4,000 Bar)

• Hydrostatic pressure chambers

• Constant pressure pump mode

• HPLC solvent compatible seals

• Column packing and separations

McCoy J., Hubbell W. L., PNAS 2011, 108(4):1331-6

HUB440 is used to pressurize fused silica capillary for SDSL-EPR of

the T4 Lysozyme mutant L46R1 to enable direct determination of

pressure-dependent equilibrium constants for studies of protein

conformational dynamics.

Pressure perturbation for SDSL protein EPR

Pressure-jump system for SDSL protein EPR

HUB 440

Pressure

Intensifier

Pressure

Sensor

Fast Valves

Reservoir

Sample Ports

Electronics

Housing

Solenoid

Computer

PBI HUB 440

Pressure Intensifier

Pressure

Sensor

Pressure

Sensor

Reservoir

Solenoid

Solenoid

PBI Fast

Valve

PBI Fast

Valve

Sample

Ports

EPR

Sample Cell

Resonator

Data: Michael Lerch and Wayne Hubbell, 2012

Future instruments

•Serial flow-through proceeding

•Parallel batch processing

•Real time spectroscopy accessories

Conclusions

• High pressure equipment offers unique and

relatively unexplored thermodynamic conditions for

life science research

• There is a largely unmet market niche for user-

friendly benchtop pressure generators capable of

dynamic control of pressure and temperature

• PBI equipment is an inexpensive modular

instrument approach designed to promote high

pressure research in multiple markets

Acknowledgements

Harvard School of

Public Health

Pproteomics

Beth Israel

Deaconess MC

Epigenetics of

Infant Nutrition

Brigham and

Women’s Hospital

Mitochondrial

biomarkers

Biopharmaceuticals

Biodefense

Cancer Biomarkers

Pathology, FFPE proteomics

Mass spectrometry, proteomics Pathology, FFPE proteomics

Structural Biology,

Biophysics

Green Chemistry

US FDA

CBER

Vaccine Development

Proteomics

SBIR-II 5R43-GM079059 and SBIR-I 1R42-AI081518

SBIR-II W81XWH-08-0247