KENYATTA UNIVERSITY

SBC SBC 370: Advanced Biochemistry

Tech. Department of Biochemistry & Biotechnology

ojolapatrick@gmail.com Dr. P. Ojola

Semester-II 2018/2018

Tissue/Cell disruption methods -Enzymes (most) thermo bile –separation need low temp eg. 4 ºC

-Disruption methods depend on cell fragility- soft one osmostic shock is enough alternating

freeze & thaw

-Enzyme digestive methods- lipases & proteases(colageanses,trrypsin, hyalurodinase or use of

organic solvents like tuluene)

-pectinases/cellulases combinations can digest Plant tissue

-Microbial cells are susceptible to lysosyme treatment

-Pestle & mortar- convenient method in presence of coarse/fine sand/ alumina

-solid shear method; not suitable for delicate animal tissue/microbial extraction

-Liquid shear;e.g in blenders with blades, appropriate for animal/plant tissue but not microbial

-homogenizers;use upward/downward mvt of ball headed plunger with bestle (power driven)

-Ultra sound thought to disrupt cell wall thro, forces in cells but heat generated may denaturation

Buffering against pH Changes in Biological Systems -every biological process is pH dependent; a small change in pH produces a large

change in rate of process

-rxns where H+ ion is a direct participant most cases

-enzymes that catalyze cellular reactions, and many of molecules which they act, have

ionizable grps with Xteristics pKa values-the logarithmic scale, the relative strength of a weak acid or base

-The protonated Amino & carboxyl grps of A.As and the phosphate

grps of nucleotides,

-ie., fxn as weak acids; their ionic state depends on the pH of surrounding medium

-Cells and organisms maintain a specific and constant cytosolic pH, ~7

-In multicellular organisms, pH of extracellular fluids is also tightly regulated.

-pH Constancy achieved primarily by biological buffers: mixtures of weak acids and

their conjugate bases.

-When conditions for centrifugal seperation of particles are reported, rotor speed, radial

dimensions & time of operation of rotor must be quoted

-Biochemical expt. Done in sol, rate of sedimentation of particle is dependent not only upon

applied centrifugal field but also mass,

-Expressed as pdt of Vol & density, density & viscosity of media and extend at which shape

deviates from spherical.

-during sedimentation , particle must displace some of solution where its suspended

-resulting in upthrust on the particle equal to weight of liquid displaced

-If particle is assumed spherical & of known Vol. & density, the latter being corrected for

buoyancy due to density of medium,

-then net outward force (F) it experiences when centrifuged at angular velocity of ῳ radians s-1

Is given by F = 4 πr 3 p(ῤp -ῤm ) ῳ2 r 3

- Where 4 πr 3p

is volume of sphere of radius rp, ῤp is density of particle, ῤm is density of medium 3

r is distance of particle from the centre of rotation.

-particle however, generate friction a s they migrate thro, soln.

-If particle is rigid & spherical moving at known velocity, then friction force ( F o) opposing

motion given by;

F o= vf v is velocity / sedimentation rate of particle, f is frictional

coeficient of particle in solvent

-frictional coeficient is fxn of it size, shape & hydration, and viscosity of mediun and by Stockes

equation, for an unhydrated spherical particle is

f = 6πȵrp where ȵ is viscosity coeficient of medium

-For asymetric/hydrated particles, actual radius of particle in equation above (immediate) is

replaced by effective or Stockes radius, reff.

- An unhydrated, spherical particle of known Vol. & density and present in medium of constact

density,

-Hence accelerates in centrifugal field, its velocity increasing till net force of sedimentation

equals frictional force resisting its motion thro, medium i.e

F=F o or 4 πr 3 p(ῤp -- ῤm ) ῳ2 r = 6πȵrp v 3

-In practice, balancing of these forces occurs quickly & particles reaches constant velocity Coz

velocity of particle

-Under such situation, net force acting on particle is zero

-hence particle no-longer accelerates but achieves max. velocity, result that sediment at constant

rate

-rate of sedimentation is given as v= dr = 2r2 p(ῤp -- ῤm ) ῳ2 r

dt 9ȵ

-this equation shows that sedimentation rate of given particle is proportional to its size, to

difference in density btw the particle & medium & applied centrifugal field

-its zero when density of particle & medium equals but decreases when viscosity of medium

increases

-since the equation involves square of particle radius, the size of particle has greatest effect upon

sedimantation rate

-Particles of similar density, but differ slightly in size can have large difference in their

sedimentation rate

-Integration of the equation above yield

t = 9ȵr ln rb

2ῳ2 r 2 p(ῤp -- ῤm ) rt

-t is sedimentaion time in seconds, rt is radial distance from axis of rotation to liquid meniscus

-rb is radial distance from axis of rotation of bottom of tube

-it is clear; mixture of heterogenous, ~ spherical, particles can be seperated by centrifugation on

basis of their densities/ size.

-Either by time needed for complete sedimentation or by extent of their sedimentation after some

time

-Considerable discrepancies exist btw theory & practical centrifugation

-Cplx variables not accounted for in last two equations like conc. Of suspension, nature of

medium & design & handling of centrifuge, will affect sedimentation properties

-Spherical particles exhibit modified relationship btw sedimentation & size, resulting slower rate

of sedimenation of such particles

-second last equation can therefore be modified to

v= dr = 2r2 p(ῤp -- ῤm ) ῳ2 r dt 9ȵ (f/fo)

-This into account the frictional effect of varying particles shape on sedimentation rate

-frictional ratio, f/fo, where f is frictional coefficient of spherical/or hydrated particle

-fo is theoritical frictional coefficient of an unhydrated sphere of same relative MM & density;

-It’s a fxn of shape & hydration of particle and for particle spherical ranges ~ 1-1.4

-hence particles of given mass but different shape sediment at different rates; this is exploited in

study of conformation of molecules by analytical ultracentrifugation

-Its convimient to consider sedimentation of particles in uniform centrfugal field, in practice it

doent occur when preparatory rotors are used

-This due to nature of design rotor

Swinging –bucket rotor

a. Cross section diagram

b. Tube initially loaded with gradient,

sample then layered on top b4

tube is placed in bucket

c. During acceleration tube reorient

perpendicularly to axis of rotation

d. Sedimentation & seperation of

particles

e. end, rotor decelerates, bucket rest to

original state

Fixed angle rotor

a. Cross section

b. Centrifuge after being filled with

gradient, is loaded with sample

then placed in rotor

c. During rotor acceleration,

reorientation of sample &

gradient occur

d. Sedimentation & seperation

during centrifugation

e. Rotor rest, gradient reorients and

bands of seperated particles

appear

Vertical tube rotor a. cross-section

b. Centrifuge tube filles with

gradient; sample layered on top

then placed in rotor

c. Rotor accelerates, sample &

gradient reorient

d. Sample & medium reorientation

complete

e. Sedimentation & separation occur

f. Reorientation of seperated

particles and gradient during

deceleration

g. Rotor rest; bands of seperated

particles & gradient fully

reoriented

-effective radial dimention of given particle change according to position in sample container

-and varries btw rmin and rmax.

-Since centrifugal force generated is proportional to ῳ2 r, particle experiences greater field

further away it from axis of rotation

-Eg. Operative centrifugal field in fixed-angle rotor can differ by a factor of 2 btw top and bottom

of centrifuge tube

-hence sedimenatation rate of particles at bottom will be twice that of identical particle near top

of tube

-hence particles will tend to move faster as they sedimenat thro, non –viscous medium

-So tis relevant to record relative centrifugal field calculated from average radius of rotation (rav)

of column of liquid in tube

i.e (distance from centre of rotation to middle of liquid column in the tube)

-average relative centrifugal field (RCFaV) is there fore numerical average of values exert at rmin

and rmax

-if sample container is partially filled , case of fixed-angle & swing-bucket rotors, min. radius

(rmin) is effectively increased

-Particle therefore start sediment in higher gravitational field with reduced path-lenght to travel

-sedimentation is quicker

-sedimentation rate/velocity (v) of particle is also expressed as its sedimentation rate /unit of

centrifugal field, called sedimentation coefficient, (s)

-from this equation v= dr = 2r2 p(ῤp -- ῤm ) ῳ2 r

dt 9ȵ (f/fo) -tis notable that composition of suspended medium is defined, then sedimentation rate is proportional to ῳ2 r, the centrifugal field, it then simplifies to v = sῳ2 r or s = v = dr/dr ῳ2 r ῳ2 r -

-Since sedimentation rate studies can be done using variety of solvent-solute systems, or different

temp,

-and expt, determined value of sedimentation coefficient (affected by temp, solution viscousity

& density) is convetionally corrected to sedimentation constant obtained theoritically in H2O

-This occur at 20ºC, and by means of equation below expressed as the ;

standard sedimentation coefficient or S20,w

S20,w = S obs x ȵT x ȵc x (1-ΰ ῤ 20,w)

ȵ20 ȵo (1- ΰ ῤT)

-S20,w is std sedimentation coefficient

-S obs is experimentally measured sedimentation coefficient,

-ȵT is viscousity of water at temp T(ºC)

-ȵ20 is viscousity of water at 20ºC , ȵc is viscousity of solventat a given temp. near 20ºC

-ῤ20,w is density of water at 20ºC

-ῤT is density of solvent at T(ºC)

-ΰ is partial specific Vol. of solute

-macromolecules eg. Nucleic acids, & proteins sedimentation coefficient decrease with increase

in conc. Of solute

-this is severe with increase in both relative MM & degree of extension of the molecule

-Hence S20,w, measured at several conc. & extrapolated to infinite dilution to get S20,w at zero

conc. Sº20,w

-Sedimentation coefficient (S20,w) of most particles are very small

-for convinience its basic unit is given as 10-13 s, referred to as Svedberg Unit (S)

-rRNA with S20,w of 5 x10-13 s, has 5S

-S20,w is influenced by shape, size and density hence commonly used to characterized particular

molecule/structure

-The larger the particle, the larger the S and hence faster sedimentation rate.

-S20,w (in S unit) for enzymes, peptide hormones & soluble proteins are 2-25 S, nucleic acids 3-

100 S, ribosomes & polysomes 20-200 S,

- viruses 40-1000 s, lysosomes 4000 S, memb100-100 x 103 s, mit.20x 103 s to 70 x 103 s, nuclei

btw 4000x 103 s & 40,000x 10-13 s

Centrifuges and use -may be classified into 4 major grps

small bench,

large capacity (refrigerated),

high speed (refrigerated), and

ultracentrifuges

belong to two type

Preparatory/analytical

Small bench

-simplest, least expensive, with many designs

-use for small qnt of material collection sedimenting rapidly (yeast,WBC,)

-max. speed 4000-6000 rev min-1 , max. RCF 3000-7000g, often operate at ambient temp

large capacity (refrigerated

-max. speed 6000 rev min-1 , & max. RCF~ 6500g

-Rotors are refrigerated vary only differ in carrying capacity (4-6 dm3 )

-can use different both swinging bucket and fixed –angle rotor

-Tube contents must be balanced to accurately; to 0.25g of each other, tubes must be even in

No.

high speed (refrigerated)

-Max rotor speed~ 25,000 rev min-1 ,RFC ~ 60 000g, total capacity 1.5 dm3

-interchangeable fixed-angle & swinging bucket rotors

-often used to collect microorganisms, cellular debris, organelle & proteins ppt by ammonium

sulphate

- Cannot generate sufficient RCF to sediment effectively viruses or smaller organelles - ribosome

Continuous flow

-a simple high speed, particles suspended in media flow continuously (~ 1-1.5 dm3 min-1)

Long tubular, non interchangeable

-Particles sediment against the wall of tube and excess overflow thro an out let

- application; harvesting bacteria & yeast from large Vol. of culture medium (10-500 dm3)

Preparatory Ultracentrifuges

-Max speed~ 80 000 rev min-1 , with RCF upto 600 000g

-rotor is refrigerated, sealed & evacuted to keep tem. generated by friction of air & rotor down

-tem. Detector is sophisticated than simple instruments with installed infra-redt emp control

-Overspeed control also available and electronic circuite to check rotor imbalance

-flexible drive shaft system to check on imbalamced due to unequal loading

-the tubes are balanced to within 0.1g of each other

-for more safety, due to speed, the rotors enclosed in heavy armour plating

-e.g air-driven, table-top (airfuge) ultracentrifuge is available, can accelerate magnetically

suspended 3.7 cm diameter rotor

-it has capacity of 6x 175 mm 3 tubes, with 100000 rev min-1 (160 000g) in ~ 30 s

-Application; biochemical &clinical research; small qnt sample requiring high centrifugal force

-e.g macromolecule/ligand binding-kinetic studies, steroid hormone receptor assay

-separation of plasmas major lipoprotein & deproteinisation of physiological fluid for amino

acids analysis

Analytical Ultracentrifuges

-Speed ~ upto 70 000 rev min-1 (500 000g), motor put in armoured plate

-Has refrigerator, with optical system to enable sedimenting particle observed for conc.

distribution determination

--are of 3 types optical system

-light absorption,

alternative Schlieren &

rayleigh

Detect changes in refractive index

of resolution

-Rotor is solid, with holes for the container, suspended from drive shaft of motor

-Thermistor , installed to measure temperature

-Several rotor types available;

a, simplest- which incorporates two cells

analytical cells & counterpoise cells (counterbalances analytical cell)

-fig 6.4b, counterpoise has two holes facilitate calibration of distance I analytical cells

-variety of cells available with capacity btw 0.4-1.0 cm 3

-analytical used with UV light absoprtion & Schlieren optical systems has single 2º or 4º sector

shape to prevent convention & has a 12 mm optical path length centre piece

a) Analytical ultracentrifuge

with Schlieren optical system

b) single sector analytical cell

& counterpoise cell

Rotor Design

Particles fan out radially &

parallel

Tube are 14º &40º to axis and

particles move radially

-pattern of sediment re-orient

Zero angle, solution re-

orient 90º, quicker

sedimentation than others

Zonal rotors

-may be of batch / continuous flow types; formers extensively used

-batch types differ in method of loading /unloading

-lows speed types operate ~ 5000 rev min-1 (5000g), high speed ~ 60 000 rev min-1 (256000g)

-body of batch-type is either large cylindrical container/hollow bowl

Elutriator rotors

-a continuous flow type with recesses to hold single conical-shaped separation chamber

-apex points away from axis of rotation & bypass chamber on opposite as counterbalance system

-seperation chamber is conical shaped so gradient of liquid flow velocity gradually decrease as

diameter of chamber increases towards centripedal end (axis of rotation)

-and will exist in chamber that opposes applied centrifugal field

-particles have different sedimentation rates in centrifugal field is balanced against controlled

flow of fluid.

-particles band in separation chamber at positions where their sedimentation velocity is

proportional to their size, balanced by liquid flow rate in opposite direction

-Larger particles accumulate towards centrifugal end where liquid flow velocity is high vise versa

-Fig.

Separation methods in preparative ultracentrifuges

Differential Centrifugation -based on difference in sedimentation rate of different size & density

-large particles sediment 1st . Those of same mass but differ in density (higher densities

eg peroxisome, ῤ=1.23g cm -3 in sucrose) sediment faster than less densities eg plasma

memb. ῤ=1.16g cm -3 in sucrose solution)

-particles with similar banding densities (most subcellular organelles, ῤ=1.1-1.3g cm -3 )

can be separated by differential or rate zonal method- if there is 10x difference in mass

-differential; material eg tissue homogenate is divided centrifugally into fractions by

increasing step wise applied centrifugal field

-Any particle originally in homogenate can be in pellet/supernatant depending on speed

& time applied and size & density of particle

-at each end, pellet & supernatant are separated & the former washed several times and

re-suspended and centrifuge at same conditions

-This minimises cross contamination, improves particle separation, with fairly pure

pellet

-Initially all particles homogeneously distributed throughout tube (a)

-During centrifugation, they move down at respective sedimentation rate (b-e) and start pellet

-pellet occurs (c) &supernatant re-centrifuged at higher speed to separate medium-sized particles

-but since various particles of differing properties were homogeneuosly distributed initially, it is

evident the sedimented particles will not be homogenous

-but will contain mixture of all sedimented components, enriched by faster sedimenting heavier

particles

-so by completion of sedimentation of larger particles, some medium-sized, originally suspended

near bottom will sediment

-this contaminate the pellet fraction; in the one step centrifugation.

-so separation is improved repeated (2-3x) re-suspension of pellet in homogenisation medium and

re-centrifugation under same previous conditions

-this will reduce the yield obtained.

-further re-centrifugation of supernatant gradually increasing centrifugal field, results in

sedimentation of intermediate & finally smallest and least dense particles

-scheme for fractionation of

rat liver

Differential centrifugation is

probably most commonly used

method for isolation of cell

organelles

From homogenised tissue

Density Gradient centrifugation

-two methods; rate zonal & isopycnic (isodensity/ equal density)

-used for determination of bouyant densities & for estimation of sedimentation coefficient

-Zonal; based on difference in size, shape & density, density & viscosity of medium and

applied centrifugal field

-but since similar types of particles often similar in shape & densities ranges small,

-and max. density of gradient chosen not to exceed that of most dense particle, to be separated,

-separation of similar type of particle therefore by zonal tech. is based mainly on difference in

size

-organelles; mit, lysosome & perixosome have different densities but similar size, don’t

separate by zonal

-seperation of proteins same densities but differing only 3 fold in RMM can be achieve by

zonal

-tech. involves careful layering of sample top of liquid density gradient, highest densities don’t

exceed that of densest particle

-gradient fxn to stabilize liquid column not to move due to convection current

-band are formed according to relative velocities of particles

-seperation terminated before particles reach bottom (time dependent)

-tech used to separate enzumes, hormones, RNA-DNA hybrids ribosomal subunits, organelles

isopycnic

-depends solely upon bouyant density of particles not shape/size and independent of time

-size affect only rate of reaching isopycnic position in gradient

-tech. used to separate particles of same size but differing densities

-soluble proteins with same densities (ῤ=1.3g cm -3 ) cannot use this method

-but organelles eg Golgi ῤ=1.1g cm -3 , mit ῤ=1.19g cm -3 peroxisome ῤ=1.23g cm -3 can

Centrifugal elutriation

-seperation & purification of large variety of cells from different tissues and species

-This can be achieved by gentle washing action using elutriator rotor

-Tech. based on difference in equilibrium in set up in separation chamber of rotor btw opposing

centripetal liquid flow & applied centrifugal field on basis of difference in size

-tech doesn’t employ density gradient

-but has advantage that any medium compatible with particles can be used

-eg. Buffered salt solutio/culture medium

-since pelleting doesn’t occur, fractionation of delicate cells/particles btw 5-50μm diameter is

achieved with ease