Unit 1: Introduction to Biophysics Biot 3109

1.1 The Role and Scope of Biophysics

Definition of the term Biophysics

•The term biophysics was first used in 1862 by Karl Pearson in his book: The Grammar of

Science.

•Several definitions of biophysics have been advanced.

Biophysics: [the fusion of biology and physics]

• is the study of biological phenomena by using physical methods and concepts;

• is not a discipline proper like genetics, biochemistry and molecular biology, but is

expected to promote interdisciplinary bridging, or

• is an advanced interdisciplinary (field of science that overlaps with other sciences)

science involving: physics, biology, chemistry, mathematics;

• is the application of the techniques, approaches, and knowledge of the physical

sciences to the problems of the life sciences;

• is a scientific field (a branch of science) widely recognized category of specialized

expertise within science, and typically embodies its own terminology and

nomenclature.

• is a natural science one that seeks to elucidate the rules that govern the natural

world using empirical and scientific methods.

• is a biological science concerned with the study of living organisms, including

their structure, function, growth, evolution, distribution, and taxonomy. Page 1

 • is a branch of physics concerned with the study of matter and its motion through

space and time, along with related concepts such as energy and force.

The Role of Biophysics

• By the beginning of the twentieth century, experimental and theoretical methods of

investigation were already used by some physicists in the study of abroad range of

problems in biology.

• Some notable researchers were:

Hugo Fricke: estimate the thickness of the cell membrane,

Nicolas Rashevsky: promoter of a Mathematical Biophysics,

Delbrck: contributions to genetics

Hodgkin-Huxley: formulation of the model of action potential propagation

Kendrew: determination of myo-globin structure

Goals of biophysics:

• Make quantitative predictions

• Experimentally test quantitative predictions

• Create simplified models of biological systems

Biological Models

[a] DNA models: [see Fig. 1]

sequence, binding site, charged rod, elastic rod, random walk

[b] Modeling transport across cell membranes: [see Fig. 2]

diffusion, active, passive transport Page 2

Fig. 1 DNA Model Fig. 2 Transport Model

[c] Protein models:

Amino acid sequence,

HP model,

Ribbon diagram,

compact random walk,

receptor,

two-state system

Fig. 3 Protein Model

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[d] Membrane models:

array of springs,

random surface,

RC circuit,

semi-permeable

Fig. 4 Membrane model

1.2 The Sub-fields of Biophysics

• The field of biophysics is already too broad to be described as a single problem or theme,

and there actually exist several sub-fields of biophysics.

• One could classify the main sub-fields of biophysics by using as criteria either the level of

organization of the living matter or the sub-fields of physics from which biophysics mainly

borrows its methods and approaches.

Based on the first criterion, one distinguishes:

1. Quantum biophysics:

investigating the behavior of living matter at sub-molecular and molecular level.

2. Molecular and supra-molecular biophysics:

sometimes called chemical biophysics and dealing, for example, with charge and energy

transfer between bio-molecules and their complexes.

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3. Cellular biophysics:

investigating single cell properties and processes (e.g., cellular excitation, membrane

mechanics, membrane structure and function, etc.).

4. Biophysics of complex systems:

e.g., neural networks, gene networks, and sensory systems.

Using the second criterion, one could speak of:

(a) Bio-mechanics:

with its sub-domains, bio-acoustics, bio-rheology, and hemodynamics

(b) Bio-thermodynamics:

e.g., bio-energetics of cellular respiration, muscle contraction and membrane transport

(c) Bio-electricity:

which investigates generation and evolution of membrane potentials, and electrical

activity of different tissues and organs

(d) Physiological optics:

investigating a wide palette of biophysical phenomena, starting with the primary physical

interaction between light and visual pigments and continuing with the interpretation of

the image by the visual cortex

(e) Photo-biophysics:

which uses physical and chemical approaches to investigate processes of photosynthesis

and bio-luminescence

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(f) Radiation biophysics:

which deals with the interaction of the living matter with ionizing and non-ionizing radiation

(g) Theoretical and computational biophysics:

which attempts to conceive new modalities of analyzing and, especially, interpreting

experimental data

1.3 Biophysical techniques and applications

• The biophysical techniques provide information about the electronic structure, size,

shape, dynamics, polarity, and modes of interaction of biological molecules.

1.3.1 Centrifugation:

Principles of centrifugation

• Centrifugation is one of the most important and widely applied research techniques in

Biochemistry, Cellular and Molecular biology, and in Medicine.

• A centrifuge is a device for separating particles from a solution according to their size, shape,

density, viscosity of the medium and rotor speed.

• In a solution, particles whose density is higher than that of the solvent sink (sediment), and

particles that are lighter than it floats to the top.

• The greater the difference in density, the faster they move. If there is no difference in

density (isopycnic conditions), the particles stay steady.

• To take advantage of even tiny differences in density to separate various particles in

a solution, gravity can be replaced with the much more powerful centrifugal force

provided by a centrifuge. Page 6

• A centrifuge is used to separate particles or macromolecules:

Cells, Sub-cellular components, Proteins, Nucleic acids

• Basis of separation:

Size, Shape, Density

• The centrifuge works using the sedimentation principle, where the centripetal acceleration

causes more dense substances to separate out along the radial direction (the bottom of

the tube).

• When particles are forced through a solution, they experience resistance to movement

which depends on properties of the particle such as mass, shape and density and

properties of the solvent such as its temperature, viscosity, density and composition.

• Thus, a commonly-used experimental format where this occurs is sedimentation in a

centrifugal field which is also called centrifugation.

• In 1926 Theodor Svedberg (1884-1971) received a Nobel Prize for his work in colloid

chemistry and invention of the new method ultracentrifuge.

Physical Basis of Centrifugation

• Relative centrifugal force:

F = M ω2 r (1)

where M = mass of particle,

r = radius of rotation (cm) (i.e. distance of particle from axis of rotation),

ω = Average angular velocity (radians/sec)

= 2π/60 (revolution per minute, rpm) Page 7

Proof of the above equation (1)

Newton’s second law:

F = ma

where F = force, m = mass, and a = acceleration

For rotational motion, the centripetal force can be given by:

𝑭𝒄 = 𝑚𝒂𝑐

But the centripetal acceleration:

𝑣2
𝒂𝑐 =
𝑟

where v = linear speed and r = radius

The linear speed (r):

v=s/t

where s = arc length (linear displacement) and t = time taken

The rotational speed (ω):

𝜽
ω= Page 8
𝒕
 where 𝜽 = angular displacement and t = time take

The angular and arc length are related by:

S = 𝜽𝐫

Thus,

𝒔 𝜽𝒓
v= = = ωr
𝒕 𝒕

Therefore, the centripetal force can be given by:

𝑣2
𝐹𝑐 = 𝑚
𝑟

ω2 𝑟 2
=m
𝑟

= 𝑚ω2 r

Note:

1revolution = 2πradians = 3600

• From Newton’s second law, force is given by:

F = mg,

where g = acceleration due to gravity

• Thus, the centrifugal Field:

G = ω2 r (2)

or for one complete cycle per minute (rpm), G can also be written as:

4π2 𝑟
G= (3)
3600

Activity
[1] Show that Eq. (2) is equal to Eq. (3).

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 𝛉
ω=
𝑡

But, for one complete cycle per minute:

θ = 1revolution = 2π rad and t = 1min = 60sec,

𝟐𝛑
ω=
60

Thus, square on both sides:

4π2
ω2 =
3600

[2]

Solution:

Activity

A centrifuge rotor is spinning at 25,000 rad/s. The ‘top’ of the cell is 5 cm from the rotor’s central
axis, and the ‘bottom’ of the cell is 9.5 cm from the central axis. What are the centrifugal fields
on a particle found at the top and at the bottom of the tube?

Interacting Forces in Centrifugation

Sedimenting force is opposed by:

F = MPω2r,
where MP is the mass of the particle Page 10
[1] Frictional Resistance against particle moving through fluid.

Force of friction= f v

where f = frictional coefficient of particle in the solvent

v = particle velocity

[2] Flotation Force

F = Ms ω2 r

where, Ms = the mass of equal volume of solvent

• The net force is equal to:

Fnet = (mp − ms)ω2 r − f v

• When the frictional force balances the driving force,

dv/dt = acceleration = 0,
The sedimentation velocity becomes v:

v = (mp − ms)ω2 r
f

= mp (1 − ρs /ρp)ω2 r
f

where ρs is density of the solvent and ρp is the particle, provided that the volume is

kept constant.

• Diffusion coefficient D, which was stated by Einstein (1906), is given by:

D = kT
f

where k is Boltzmann constant and T = temperature.

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• Hence,

v = Dmp (1 − ρs / ρp )ω2 r
kT

• Sedimentation velocity is dependent on the shape of the particle but, for a perfect sphere
(f = 6πrη - Stokes law and mp = ρp Vp but Vp = 4/3πr3 ), this quantity is related to physical
properties of the particle and solvent by;

v = mp (1 − ρs /ρp)ω2 r
f

= ρp Vp (1 − ρs /ρp)ω2 r
6πrη

= 4/3πr3 (ρp − ρs )ω2 r

6πrη

= a2 [ρp − ρs]ω2 r
18η

where a (2r) is the particle diameter, ρp and ρs are the densities of the particle and solvent,
respectively and η is the viscosity of the solvent.

• The inverse relationship with viscosity means that sedimentation velocity decreases markedly
with increase in solvent viscosity and (since viscosity is dependent on temperature) with
temperature.

Sedimentation Coefficient (s):

•A useful measure of differential behaviour in a centrifugal field is provided by the sedimentation

coefficient, s:
s= v
ω2 r
= (mp − ms)
f Page 12
 = M (1 − ρs / ρp)
NA f

where M is molar mass and NA is Avogadro’s number

Since both mp (1 − ρs /ρp) and f are inherent properties of the particle and solvent, respectively, s
is a constant for any given particle/solvent.

• Note:-
1) Factors affecting s are molecular weight, frictional force and solute.
2) S is increased for particle of larger mass
3) S is increased for particle of larger density (equal volume)
4) S is increased for more compact structures (Shape) of equal particle mass (frictional

coefficient is less)
5) S is increased with rotational speed
6) The range of values observed in biochemistry varies widely from as low as 1x10 −13 sec

for a protein to 10000x10 −13 sec for intracellular organelles.

• Applications

Separation of biological molecules i.e, bio-molecules on basis of density like DNA, RNA.

Separation of sub-cellular organelles-Chloroplast

Separation of labelled and non-labelled molecules.

Separation of ribosomal subunit given as Svedberg units

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Activity
Q] Estimate how long does it take to settle a protein by 1 cm in
a) low speed of rotation, ω ∼ 1000rpm , and
b) high speed of rotation, ω ∼ 100, 000rpm

(mp ∼ 10−27 kg, D = 10−11 m2 /s, k = 1.38x10 −23 J/K, T = 273K, r = 10cm, ρp = 1.36986g/cm3 and
ρwater = 0.9982 g/cm3)

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b) Electrophoresis

•To separate molecules of different molecular mass/size based on the sedimentation

principle.

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• The coulomb force Fel, due the electric field acts on the charged molecules, can be

given as:
Fel = EQ,

where E - electric field and Q - particle charge

• As the particle passes through the solvent it experiences resistance due to a frictional

force,
F∗ = fv

Thus, the particle velocity is given by:

v = EQ
f

• Electrophoretic mobility u is written as:

u=v/E=Q/f

• Diffusion coefficient D, which was sated by Einstein (1906), is given by:

D = kT / f

where k is Boltzmann constant and T = temperature.

• Hence,

u = QD
kT

Note:

Gives poor results due to complicated friction mechanism for ions: counterion and solvation
shell, shell deformation at higher velocities.

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c) Size Exclusion Chromatography (SEC)

•uses tightly packed gel beads and sedimentation based on gravity (and sometimes pressure) to

trap small molecules and allow larger molecules to pass through the gel faster than small

molecules.

d) Spectroscopy mostly

• with incident EM radiation and measuring the intensity /direction /polarization of the emitted

radiation (originally only the visible spectrum 380750 nm was used; now also UV and IR); in

addition to EM also electron and mass spectroscopy.

e) Absorption Spectroscopy

• to find e.g. the concentration of molecules in the solution by using EM of a particular to shine

on the sample and measure the intensity that comes out OR absorbance versus to identify the

type of molecules.

f ) Fluorescence Spectroscopy

• to characterize molecules and to follow conformational transitions; caused by absorption

at a one wavelength and emission at a longer wavelength (electrons drop from their excited

energy state emitting light.

g) Mass Spectrometry

• to measure mass or molecular weight of molecules; molecules are ionized in a vacuum,

then passed through a magnetic field.

h) X-Ray Crystallography

• to determine the relative positions of atoms within a crystal by using diffraction on a 3D crystal

lattice; high resolution of structural details but the molecules need to in a crystalline phase.

i) Nuclear Magnetic Resonance Spectroscopy (NMR)

• to obtain structural information about molecules of the highest resolution using EM of a radio

frequency, which interacts with nuclear spins of atoms in a large magnetic field,

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 causing them to jump between the spin states and emit at different depending on the local

structure around the atom.

j) Electron Microscopy

• to view objects 1,0002,500 smaller than those seen by light microscopes (electrons of a small

wavelength are used instead of EM); transmission EM (TEM) and scanning electron

microscopy (SEM).

k) Atomic Force Microscopy (AFM)

• with resolution similar to TEM, 3D features like SEM; a mechanical probe (a tip)

moves along the surface of the scanned object to obtain 3D information.

l) Optical Tweezers

• to hold and manipulate microscopic particles even single molecules or atoms using

focused laser beams to create forces of the order of pN = 10 −12 N (0.1 nm to 10,000

nm size objects) and measure forces needed to bend or break DNA, for example.

m) Voltage Clamp

• is used in electrophysiology to determine electric currents in cells, in particular neurons; a

fine microelectrode is inserted into the cell with another in contact with the surrounding

fluid while the voltage is clamped (held constant) by feedback that generates a

countercurrent to that generated by the cell.

n) Current Clamp

• is analogous to voltage clamp; the current is clamped (held constant) and the voltage

change induced by the cell measured.

o) Patch Clamp

• is alternative to voltage/current clamp; the electrode is placed inside a micropipette with

electrolyte solution and the micropipette combined with a gentle suction electrically

isolates a small patch on the membrane; enables to study a single ion channel within the

membrane. Page 19
p) Calorimetry

• measures C or C versus T: transitions or ligand binding.

Worksheet for Unit One

[1] Explain briefly about the purpose and applications of centrifugation and Electrophoresis.

[2] A particle with a charge of 1μC is moving at a speed of 2x106 m/s in a solvent under the

application of uniform electric field of strength 200N/C. Determine the frictional coefficient

of particle in the solvent and the electrophoretic mobility.

[3] Show that the sedimentation velocity of a particle is zero, where its density equals to the

density of the medium.

[4] Based on the given numerical date, determine the ratio of sedimentation velocity of O2 to

glucose. (Use: ω2 r = 106 m/s2)

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