Spread monolayers of proteins

Advances in Colloid and Interface Science, 25 (1986) 341-385

341

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

SPREAD MONOLAYERS

OF PROTEINS

F. MacRITCHIE

CSIRO Wheat Research Unit, P. 0. Box 7, North Ryde, N.S.W. 2113, AUSTRALIA

CONTENTS I.

ABSTRACT

.......................................................

II.

INTRODUCTION

III.

METHODS

...................................................

FOR SPREADING

A. Aqueous

solution

B. Spreading

for quantitative AND INTERPRETATION

0. Surface

potentials

B. Surface

rheology

346

....................

347

films

.................

MONOLAYERS

...............

349 350 352 355

..........................................

355 356

of film ............................................

358

methods

......................................

362

.....................................

363

........................................

363

FROM MONOLAYERS

A. Kinetic measurements B. Radiolabelled C. Desorption

proteins

......................................

from mixed monolayers

0. Effect of molecular

weight

............................

..................................

367 369 370

E. Theory of desorption

........................................

371

IN MONOLAYERS

........................................

372

.............................................

373

REACTIONS

A. Instrumentation B. Enzyme reactions C. Other reactions

VIII.

346

...................

......................................... PROTEIN

345

............................................

0. Direct optical

VII.

weight

in compressed

FOR STUDYING

A. Surface

DESORPTION

OF n-A CURVES

..........................................

changes

coagulation

.........................

spreading

region and molecular

of state

OTHER TECHNIQUES

C. Transfer

VI.

345

0. Criteria

C. Configurational

344 344

MEASUREMENT

B. Equations

342

..........................................

....................................................

A. Low pressure

V.

...............................

............................................

solvents

C. Crystals

IV.

MONOLAYERS

342

APPLICATIONS A. Biological

and biological

activity

....................

.............................................

...................................................

376 378

........................................

378

B. Foams and emulsions

.........................................

379

C. Biomedical

.........................................

380

0001-8686/86/$15.75

membranes

374

problems

0 1986 Elsevier Science Publishers B.V.

342 IX.

CONCLUOING

REMARKS

X.

REFERENCES

.....................................................

I.

.............................................

383

ABSTRACT The study of spread monolayers

the fundamental

behavior

from their ubiquitous proteins

of proteins

presence

liposomes

arrangement and packing

trolled.

Methods

potential

of monolayers

spectroscopy,

changes,

ellipsometry).

molecular

surface

theoretical reviewed.

relevance

of multicompartment

to biological

occurring

where

the enzyme

of monolayer

membranes,

region

interface

and is

has proved valu-

This instrumentation

at the surface,

is imnobilized

for

reflectance

Experimental

film balances

in monolayers.

foams and emulsions

diffrac-

methods

configurational

from the air/water

studies are briefly

removal

in the low pressure

are all observed.

of proteins

to the study of enzyme reactions

to reactions

Some applications

(e.g., multiple

and desorption

able for the study of reactions has been applied

electron

At higher pressures,

work on the desorption The introduction

(such as surface

Direct optical

The use of measurements

that

and con-

including

is discussed.

weights

coagulation

methods

techniques,

in situ are also available --

In

chemistry.

used for their manipu-

innovations,

are discussed.

of

to other branches

may be measured

and techniques

more recent

spectroscopy,

mimetic

the unique advantage

in monolayers

proteins

for study by radiotracer

(IR)

the study of monolayers

to measure

of molecules

despite

resulting

Spread monolayers

in comparison

As well as the more traditional

on slides

tion and infrared

in nature.

field of membrane

neglected

and viscosity),

for understanding

as well as the many phenomena

and vesicles),

for spreading

lation are outlined. pressure,

is of interest

at interfaces

it has been somewhat

(such as bilayers, the

of proteins

is a branch of the developing

recent times,

II.

381

of direct

in the cell membrane.

illustrated

and biomedical

with reference

problems.

INTRODUCTION A striking

solution,

property

of proteins

is that, although

they may be spread quantitatively

to give highly stable monomolecular pounds, which

require

at these interfaces. results mainly large size. attached

from two properties:

A protein

molecule

and glutamic

a configuration

or oil/water

This is in contrast

solubility

1) their polar/non-polar

consists

ofa polypeptide degrees

and leucine)

acids).

to most com-

at interfaces

nature;

backbone

of polarity,

and 2) their

to which are

ranging

from

to polar ones (such as the

In the dissolved

that minimizes

in aqueous interfaces

in order to spread as monolayers

of protein molecules

of varying

(such as phenylalanine

aspartic

cules assume

films.

The great stability

amino acid side-chains

very non-polar ionizable

very low water

many are soluble

at air/water

state,

protein mole-

the free energy of the system.

Thus,

343 they fold up in such a manner groups at the periphery side, protected

that the number of polar

are maximized

from energetically of non-polar

unfavorable

Complete

shielding

however,

because

residues

sequence

and the steric constraints

of their possible

When a molecule

reaches

proximity

order

for this to occur,

conformation

in which

the non-polar

is replaced

to polar residues

has a certain of the molecule

segments.

Although

each segment

adsorption

(AG), the total free energy

if the number of segments

stability quires

The drastic

unfolding

molecules

the effects

has only a moderately

to an insoluble

priate.

interaction

on proteins,

large free energy of

However,

systems,

in response

to changes

interfaces.

to many biological

phenomena

produce

coagulation

in their energy

that,

of proteins that occur

appears

are continually

inappro-

altering

environment,

their

including

with interfaces

(such as cell membrane

to

irrever-

processes

denaturation"

It is this interaction

of

being applied

by the observation

when the different

re-

from the interface.

loss of solubility

are shaken,

protein molecules

very high

for the great

of a whole molecule

that surfaces

the term "surface

toward

This ex-

so that for many pur-

This accounts

by an apparent

implying

phase.

(nAG) may attain

the desorption

solutions

form results.

with

per molecule

This view is supported

protein

are understood,

In biological

conformations

accompanied

In chain

in terms of a series of

of a large number of segments

in proteins.

cases where

at interfaces

relevant

may be described

has led to the term "surface denaturation"

of surfaces

sible changes in certain

removai

directed

to the aqueous

(n) is large.

interface

side-chains.

into a two-dimensional

degree of flexibility

of protein monolayers,since

simultaneous

protein

unfold

protein

side chains are predominantly

poses, the behavior

is encountered.

if an area of high energy

by non-polar

phase and the polar side chains

tended configuration

values

medium.

achieved,

in the chain

a new energy environment

protein molecules

non-polar

not be closely

in-

imposed on chain folding.

an interface,

by water molecules

ionizable)

groups are mainly

contact with the aqueous

can usually

The free energy of the system may be reduced occupied

(especially

and the non-polar

that is

structure

and

created

model

function). A growing systems

field of research

is concerned

of cells and cell membranes.

This has become

chemistry

(ref. 1,2) and includes monolayers,

liposomes

and vesicles.

of the film balance, attention

with artificially

Despite attracting

(such as bilayers,

times.

This does not mean that there

Present

techniques

are not capable

3.4) has drawn attention

is no

multilayers,

much interest

the study of protein monolayers

as other areas

known as membrane

bilayers,

micelles

following

develapment

has not received and liposomes)

mimetic

micelles,

as much

in recent

scope for further work or that

of yielding

to the use of designed

new information. monolayer

tibius

assemblies

(ref.

to create

344 processes

analogous

cules

in biological

which

light energy

cific proteins

to those performed systems.

is converted

assemblies

of biological

monolayer

research

advantage

over some of the complementary may be manipulated

a function

determined

METHODS Although

FOR SPREADING

the technique

tional

monolayers.

are adsorbed. proportion

These conditions

in incomplete

proteins.

A. Aqueous

solution

Purely aqueous

solution

precautions

in which

A method

is allowed

satisfactory

of all protein molecules

Rigorous

theory was applied

variables

fully by a number of workers spreading

solvent

theory,

to adsorption

difficult solution

to derive appropriate

into the system. technique

This may be explained

to the surface

ionic strength, satisfactorily.

the electrical

energy

conditions

thickness

in

in terms

of flowing

aque-

has been used successthat no non-aqueous to be occas-

of energy barriers

Proteins

of mole-

are particularly

charge and the spreading

spreading

solution

and sub-

point of the protein and at a moderate

barrier

In fact, some proteins

glass

the elapsed

is to prevent a fraction

By having

strate at a pH close to the isoelectric

(ref. 5)

does not work as well as predicted

from adsorbing.

electrolyte.

for

unless

wetted

There do appear

to spread when they carry a net electrical does not contain

by Trurnit

by the presence

(ref. 6,7), the effect of which

cules which diffuse

are available

to the surface during

This method of spreading

is introduced

so that if a

then becomes a problem

and has the great advantage

ions, however, when this spreading by diffusion

spreading

(such as protein concentration,

ous film and length of rod).

of

or be convected

to flow down a perfectly

ensuring time.

spreading

for spreading

has been devised

surface.

of relevant

Quantitative

it will diffuse Several methods

is not usually

solution

quantitative

in which all the protein molecules

rod onto the water diffusion

as

is not as simple as that of conven-

are not always easy to achieve

spreading.

are taken.

an aqueous

chem-

measured

concentrations.

The secret for obtaining

is to create conditions

spreading

mimetic

properties

stable once they become attached

for spreading

of the protein does not adsorb,

away, resulting

special

two-dimensional

for furin protein

have an important

of membrane

and various

are extremely

to an interface, insoluble

promise

MONOLAYERS

protein molecules

protein monolayers

Monolayers

in

The use of spe-

that a resurgence

techniques

istry in that molecules

III.

holds sufficient

mole-

membrane

stored energy.

processes

seems likely to eventuate.

of precisely

arrays of cooperating

is the photosynthetic

into chemically

in such organized

thering our understanding

by complex

One example

is reduced and many proteins

may be spread quantitatively

spread

under these

conditions

simply by using a microsyringe

example,

bovine

solution

of pH 7.3 containing

composition

serum albumin

spread quantitatively

useful

B. Spreading

efficiency

Welcome) tative

solvent with

remain which

of isopropanol-water by Stallberg

is usually

monolayers

stable

and Teorell

is present

dissolve

the protein

are also soluble, may be spread.

in this solvent initially

vent at low temperature, become turbid,

Another

or are slowly in aqueous

ensuring

this may be avoided

solutions.

rapid mixing.

However,

clear,

ing ionic strength

concluded strength

and alcohol

of spreading

0.01, containing

Some proteins

concentration

solution

0.5% amyl alcohol

do not

It is preferable

the resulting

solution

solution

furof

as "denaturing the solutions is identical,

solution.

Small quanti-

in spreading

of different

of the spreading

to

sol-

are skeptical

that, providing

the effects

of human serum albumin

that the ideal spreading

contamination

of spread monolayers

have also been used to assist

lipids

protein-

is that the solutions

Some workers

or purely aqueous

et al. (ref. 12) have studied

quanti-

and then add the mixed

Should

experience

the behavior

spread from isopropanol/water

ties of isoamyl alcohol

mixed

because of their reputation

it is the author's

perfectly

(Burroughs

that certain

the pH of the aqueous

solvents

as protein

advantage

solution

by moving

purpose

With this solvent,

precipitated.

point of the protein.

efficiency

could

0.5 M sodium

the danger of bacterial

using alcohols

Minones

proteins

so that homogeneous

ther from the isoelectric

whether

do not

a more detailed

(ref. 9) as a general

is used.

in the case of purely aqueous

easily

are prepared

of the same

or "Agla" microsyringe

It has the advantage

achieved.

for long periods without

dissolve

agents".

for different

(60:40) containing

A micropipette

for proteins.

phospholipids)

phospholipid

This suggests'that

of spreading

its glass tip at the surface

spreading

(including

on a substrate

(e.g., bovine v-globulin)

solvents

was introduced

spreading

proteins

spread from a buffer

information.

A solvent consisting acetate

other

under these conditions.

study of the comparative yield

0.15 M sodium chloride

However,

(ref. 8).

For

with the tip in the surface.

(BSA) has been quantitatively

(ref. 10,ll).

variables solvent)

(HSA) monolayers.

(includ-

on the

These authors

is a buffer of pH 5.1 and ionic (v/v).

C. Crystals Many proteins convenient spreading constant

method

spread

rapidly

is not required surface

cult spreading

from crystals

to use for certain

purposes,

(such as where

pressure),

from solution

the method

placed at a surface. For example,

kinetic measurements

is rapid, avoiding

and the use of spreading

This is a

if quantitative are to be made at

the problem of diffi-

solvents.

In theory,

it

346 can also be adapted capable

for quantitative

of measuring

of protein

microgram

has a surface

spreading

quantities

Losses of protein

ever, the surface of dissolution,

concentration

D. Criteria

by dissolution

spreading

with a balance

A close-packed monolayer -2 in the order of 1 mg m . Therefore, to

spread a film of half this concentration Pg.

in conjunction

(ref. 13).

on an area of 400 cm2 would

require

into the bulk phase can also occur.

rate for many proteins

is much greater

20

How-

than the rate

so that losses are often minimal.

for quantitative

spreading

Since a large number of proteins which extrapolate rough indicator

give surface pressure (rr)-area (A) curves -1 to an area close.to 1.0 m'mg , this value can be used as a

of quantitative

than this almost certainly curred.

spreading.

A calculated

means that a loss of protein

If there is doubt about the efficiency

between

replicate

is to use several

spreadings spreading

Losses will usually concentration

of spreading,

is not a useful criterion. solutions

increase with

of different

increasing

of about 0.03% is recommended

protein

that the film is not under any appreciable completion

is a barrier

to the adsorption

the surface

against a surface where rier.

of spreading.

the monolayer However,

When protein

a different

n-A curve

MEASUREMENT

caused

AND INTERPRETATION

When a monolayer of the clean is defined

Vaiues of II are usualiy change

in interfacial

measurement

expressed

for monolayers.

and for many purposes

(or interfacial)

free energy

during and

pressure,

there

of the molecules

is spread in this way from the one

by compressions

with a bar-

a surface

pressure.

OF n-A CURVES

(y,) may be reduced

as the surface

It is

by the failure to take into account

is spread at an interface,

interface

pressure

a surface

is obtained

is spread at high area followed

this is an artifact

A protein

solution.

step (ref. 6), so that a fraction

the losses of protein when spread against

IV.

concentrations.

surface

When spread against

is not adsorbed.

pressure,

close agreement

for the spreading

important

smaller

has oc-

The best way to check

protein concentration.

following

reaching

area appreciably by dissolution

the initial

to a new value pressure

interfacial (y).

tension

The difference

(II); i.e.:

in mNm-' and are numerically equal to the -2 . II is thus the most fundamental

in mJm

Its units are those of a two-dimensional

it is analogous

to the more comn

pressure

three-dimensional

pres-

sure. Protein monolayers which the monolayer

may be easily manipulated is compressed

or expanded

by means of a film balance between

movable

barriers

or a

ln

341 flexible

ribbon and relations Interfacial

(n-A curves).

plate or a Cangmuir

between

pressure

interfacial

pressure

may be measured

float connected

to a torsion

and area established

by either a Wilhelmy

wire or force transducer.

main problem with the Wilhelmy

plate method

is the maintenance

angle.

angle caused

by spreading

Changes

in the contact

by the use of isolation appeared

(ref. 14).

clean glass,

holding always

mica or platinum)

it over a smoky Bunsen keep the plate wetted

elasticity. on either

Therefore,

particularly automatic

flame is convenient

A. Low pressure

to calculate

weights

remains

ecular weights

of small

Early workers

in composition

is more susceptible

Many film balances

leading

available

film balance

that employs

to leakage,

are arranged

for

(MCN Lauda, Germany

the iangmuir

float principle.

weights at very low values of n (below 0.1 mNm-' caused many workers More recently,

one for determination

to moderately

sized proteins.

phase transitions

analogous

in the field to

interest

has dwindled

of number-average

mol-

with other

In comn

in protein monolayers.

to the gaseous-type

monolayers

is not observed.

Nevertheless,

at sufficiently

act independently

and therefore

n-A relations

a colligative

high visco-

by dual barriers

to keep the film uniform

(ref. 15).

a legitimate

a region completely

molecules

compression

of proteins

there are no well defined

protein molecules reflect

instrument

on this region

but the method

(such as

be taken to

exhibit

of the plate from the vertical,

but this method

A commercially

molecular

but care should

Protein monolayers

pressures.

use of n-A measurements

focus attention

simpler

angles

regilon and molecular

The possible

Therefore,

tilting

at high interfacial

automated

has dis-

In the case of the Langmuir float, there are no prob-

contact

operations.

plate

Where the lower phase is an

a film balance which allows

measurements.

is a reliable,

polymers,

is used.

side of the plate is advisable

lems with changing

can be avoided

A mica plate coated with lamp black by

by the oil.

over the surface and to prevent to incorrect

solvents

which can be removed after the solvent

plate may be used.

The

of a zero contact

With water as the lower phase, a hydrophilic

roughened

oil. a hydrophobic

barriers

hanging

of

high areas, in this region

property.

(ref. 16) applied

an Amagat-type

equation

for imperfect

gases;

i.e.: n(A- Ao) = nRT

,

(2

where A is the area available Universal

gas constant

that the surface

aratingthe

and T the absolute

pressure

to a two-dimensional

temperature..

Bull

in this region was thermodynamically

osmotic

film-covered

to n moles of film, A, is a constant,

pressure

surface

than to a gas pressure.

R is the

(ref. 17) argued more equivalent The barrier

from the clean surface may be considered

sep-

as a

1

348 semi-permeablemembrane. A similar equation to Eq. 2 is obtained from which a plot of flA vs. i'l (extrapolatedto zero n) may be used to evaluate the molecular weight (M) from the relation:

(n A),,0 = ; RT

3

2 -1 where w is the weight of monolayer substance. If II is in mNm-l, A in cm g , then w is unity (1 g), R has the value 8.3x lo7 ergs per mole per degree and -2 T is the absolute temperature. Since A is normally expressed in mgm , this value must be converted to cm 2g-' by multiplying by 107. Molecular weights of many proteins (ovalbumin, gliadin, hemoglobin, f+lactoglobulin)calculated by the film balance method agree well with values obtained by other methods (ref. 15). One of the most thorough studies was carried out by Harrap (ref. 11) on the molecular weight of insulin over a wide range of pH and ionic strength. Some of the results are illustrated in Fig. 1 for a

subsolution of pH 2.05 and

differing ionic strengths.

Fig. 1. n-A and nA-n for insulin films spread on subsolutions of pH 2.05 and differing ionic strength. Curve a: r/ = 0.01; b: r/2= 0.1; c: r/ = 0.2; d: r/2= 0.5. Some values of Fredericq (ref. 1i ) (0) are also shown for t e range in which the two sets of data overlap (ref. 11).

2

At the lowest ionic strength (f= O,Ol), the nA-n plot is linear at all values, leading to an extrapolated value of 6,000 for the molecular weight of insulin.

349

At higher ionic strengths, the curves are convex to the IIaxis.

This behavior

may be compared to PV-P curves of real gases and similarly indicates aggregation due to the tendency of attractive forces to predominate over the electrical repulsion. Extrapolationof the linear portions in these cases thus gives false values for the molecular weight, although at sufficiently low pressures, the limiting value. Effects of aggregation

curves are seen to approach a comn

at a given ionic strength were greatest at pHs

near the isoelectric point (pH

5.6) and were absent only when the molecule carried a high net charge as for the example in Fig. 1 (pH 2.05, f 0.01). This work emphasizes the advantage of measuring molecular weights under conditions where the protein carries a high net electrical charge,

If this is not possible, measurements must be made

at very low surface pressures (e.g., below 0.02 mNm-' for the results in Fig. 1). Under these conditions, a very sensitive and stable instrument is required. Extremely clean water, free of surface active impurities, is essential for this work.

Providing these considerations are observed, the method is a rapid and

reliable one for estimating molecular weights of proteins. B. Equations of state An equation such as Eq. 3 is strictly valid only for molecules which behave as rigid disks. However, protein molecules in monolayers are unfolded and the extended chains have a degree of flexibility, A number of equations have been developed to describe linear polymers at interfaces by application of statistical thermodynamics. One of the best known,is the equation of Singer (ref. 19) based on the high polymer theories of Huggins (ref. 20) and Flory (ref. 21).

Singer's equation for a completely unfolded monolayer is:

rl=J$lnII-$\+$Q-$

(,I - 2A 1

iln-+

,

where t is the total number of segments per molecule, A is the area per segment available, A, is the limiting area per segment and z is the surface coordination number in the two-dimensionalquasi-lattice in the interface. For a completely rigid chain, z= 2 and for a completely random chain z= 4. Singer's equation predictsthat if z is appreciably greater than 2, curvature in the plot of IIA vs. IIoccurs due to entropic effects arising from the random arrangements and flexibility of the long chains, For very dilute monolayers, the equation has been found to show good agreement (ref. 22). Other contributions to equations of state which take into account surface activity coefficients (ref. 23), partial solution of polymer chains in the substrate (ref. 24) intermolecularcohesion (ref. 25) and electrical 'intermolecularrepulsion (ref. 26) are relevant to protein monolayers.

350 An equation of state of the virial type (ref. 27) has been applied to x-A measurements of several proteins (ref. 28). The data were interpreted to mean that the proteins BSA, hemoglobin and ovalbumin were more unfolded than transferrin and myoglobin. By measuring changes in n-A curves as a function of temperature and applying a suitably modified Singer equation, Llopis and Albert (ref. 29,30) have derived thermodynamic spreading terms for protein monolayers. Spreading entropy (S,) and enthalpy (H,) were plotted as a function of the area and were shown to depend markedly on the pH of the substrate. The spreading terms were in accord with the notion that cohesive forces play a more important role in y-globulin than in BSA monolayers. C. Configurationalchanges in compressed films When protein monolayers are compressed to higher pressures than those considered in the previous section, relaxation processes occur which are manifested by losses in surface area (if n is kept constant) or losses in surface pressure (A constant). Providing there is no desorption'(Part VI) or surface coagulation (Part IV-D), these losses are recoverable on expansion. The very large reversible reductions in area that are observed at high pressures (II>20 mNm_') can only be rationalized if it is assumed that parts of the molecules are forced out of the surface. It has been well established that adsorbed linear polymer molecules may exist as trains (segments attached to the surface), loops (segments extending into the adjacent phase) and tails (segments at the ends of molecules) which are also preferentiallydisplaced from the surface (ref. 31, 32). Monolayers allow the opportunity to study the distribution of attached and displaced molecular segments. In the case of proteins, the relaxation processes are often sufficiently slow to enable them to be easily followed experimentally. If A, is the initial area of a protein monolayer before any relaxation has occurred and A is the area after equilibrium has been established between adsorbed and displaced segments at a constant pressure II,then the ratio of adsorbed to displaced segments (r) is given by:

If the work required for a displaced segment to enter the surface is IIAA, (ref. 33), where AA, is the area per segment, then the variation of r with IIwill be given by:

(AG, - rrAA,) r = exp kT

,

(6)

351 where AGO is the difference ments at n=O.

in free energy between adsorbed and displaced seg-

Plots of log r vs. n for monolayers of catalase at several values

of pH are shown in Fig. 2.

Fi 2. Log r vs..n for monolayers of catalase on buffer solutions at 23'C: CopI pH 7.3; (0) pH 4.4; (Cl) pH 3.5 (ref. 33). The linearity of these plots are in agreement with the premises of Eq. 6. From the slopes and intercepts, hGs and &A, values may be calculated. A summary of these values for several proteins under different conditions of temperature and substrate pH is given in Table 1. Values of CAM fall In the range 0.9-1.5 nm2, correspondi~ngto an average size of 6-10 amino acid residues per segment, hGS values fall in the range 4,6-8,8 kT per segment for the proteins studied. In general, AG, decreases with IncreasIng electrical charge as the substrate pH moves away from the isoelectrjc point of the protein (i,e., the equilibrium distribution shifts in favor of the di,splacedsegments). This reflects a contribution to aG, of an electrical term of the form q,$, where q, is th, electrical charge per segment and J, Is the electrical potential in the plane of the charged groups. On the other hand, increasing temperature increases AG, in the range studied, favoring the adsorbed segments, Thls result indicates that adsorption of segments is accompanied by an @crease of entropy, Disordering processes that may be involved are the elPminatlon of ordered water around non-polar side chains and loss of secondary structure (e.g., o-helix) by the protein.

352 TABLE 1 Values of AG, (II= 0) and AA, for protein monolayers (ref. 33). Protein

Subphase

T-Globulin

buffer, buffer, buffer;

T-Globulin

distilled

Catalase

Ferritin

Because

Temp. (OC)

pH 7.3 pH 4.4 pH 3.5 water,

distilled

pH 5.4

protein monolayers,

water,

configurational

the most meaningful

the equilibrium equilibrium, quires

distribution

although

long periods

fore, when-film recording

equilibrium

n-A curve

construct

constant

represent

curve

the n-A relations

D. Surface

;55 25

7.7 8.8 8.2 7.5

kT kT kT kT

1.5 1.5 1.6 1.5

25

7.8 kT

1.2

occur relatively

is probably

and displaced

segments

is shown

(above 20 mNm-l).

rapid stepwise

of the essentially

There-

with automatic

An example

of an

Here it is compared

compressions

unrelaxed

re-

that these measured

parameters.

in Fig. 3.

from a two-dimensional

of monolayers

some study (ref. 35).

is an interesting

are made to

monolayer.

Theoretically,

to a three-dimensional phenomenon

a monolayer

to its bulk state) on reaching

pressure

(E.S.P.).

In certain

state when the E.S.P. energy barrier

cases, monolayers

is exceeded.

associated

This

with creation

occurs

because

spreading

in a supersaturated

of the need to overcome an

energy

in crystal

nuclei

(ref. 36). proteins

spread

from their crystals

true E.S.P.s are obtained

(Part

and none appear

to

should begin to

its equilibrium

may remain

of surface

state on com-

which has been subjected

substance

(transform

Although

of

This

pressures,

rates of compression

collapse

whether

in

the equilibrium

at lower surface

fundamental

in which

slowly

coagulation

The transition pression

employing

1.4

25

at higher pressures

for a protein

with an instantaneous

4.6 kT 5.1 kT 5.8 kT

of n-A curves are used, it needs to be realized

curves do not necessarily

1.3 1.3

point corresponds to the attainment

fairly rapidly

(many minutes)

balances

7.3 kT 6.4 kT 5.5 kT

255 45

changes

between attached

reached

s2 (nm 1

0.9 1.0 1.1

II-A curve

one (i.e., the one in which each measured

AA

(n=O)

pH 5.4

pH 5.4

conditions

AGS

25 25 25

distilled water, buffer, pH 7.3 buffer, pH 4.4 buffer, pH 3.5

these reversible

under different

III-C),

it is doubtful

to have been reported

for

363

I

I

100

50

equilibrium curve; Fig. 3. n-A curves of BSA on distilled water at 2o"c; -: ----: curve which would be obtained if no expulsion of segments from interface (ref. 34).

pure proteins. by surfaces,

Because

proteins

ties to the crystals solutions

of the drastic

form a coagulum

protein.

extent of coagulation strength.

conditions,

structure

induced

and proper-

This is seen when protein

often resulting in turbidity

in the formation

of solutions

of a

on shaking was made by Henson et al. (ref. 37).

during a one-hour

Of the thirteen

coagulation

molecules.

A study of the increase

large range of proteins

of protein molecules

with very different

formed by globular

are shaken under certain

of insoluble

unfolding

proteins

and a-lactalbumin

shaking

examined,

period varied with pH and ionic

ovalbumin

was least susceptible.

globin and pepsin are other proteins

The

was most susceptible

s-lactoglobulin,

that are relatively

to

hemo-

easily surface

coag-

ulated. A simple theory protein

a substance surface

has been proposed

monolayers

will remain stable as a monolayer

free energy,

the surface by splitting Ya=Yb+Yab

ya, and will continue

free energy

in the presence

it into three terms, + nc

provided

stability

films (ref. 38). it lowers

of

Briefly,

the initial

to spread until ya= ye, where ye is

of the monolayer.

ye may be interpreted

such that at equilibrium:

(6) free energy

and the non-aqueous

pressure.

between

the non-polar

phase, Tab is the interfacial

the polar groups and the aqueous sure or coagulation

governing

to duplex

,

where yb is the interfacial monolayer

for the conditions

based on their resemblance

groups of the

free energy

phase and II~ is the equilibrium

between

spreading

pres-

354 This equation predicts that once this critical surface pressure (II~)is reached, whether by compression of a monolayer in a film balance or by adsorption from solution (as occurs during shaking), transformationof the protein to an insoluble coagulated form will occur. The results of Henson et al. (ref. 37) confirm that spontaneous surface coagulation is frequently observed when protein solutions are shaken, this process creating large extensions of interface. Certain results agree with Eq. 6, at least semi-quantitatively. This is illustrated by some experimental measurements of flc(for ovalbumiln)as summarized in Table 2. TABLE 2 Measured coagulation pressures and separated free energy terms for ovalbumin at different interfaces (20°C) (ref. 38). Interface

ya (mNm-l)

%

(mNm-I)

'b (mNm-I)

Yab (mNm-I)

Air/water

72

27

35

10

Air/5M NH4Ac

67.5

17

35

15.5

Heptane/water

50

16

24

10

Benzene/water

35

12

13

10

For example, II~is lowered as y, decreases, so that coagulation occurs more readily at O/W than at A/W interfaces. Values of yb and Tab were calculated from contact angle measurements on Langmuir-Blodgettfilms removed from a closepacked ovalbumin monolayer, The differences in surface coagulation behavior between proteins (ref. 37) must reflect variations in composition or structure which can be interpreted in terms of the yb and yab terms. These rather large differences are observed despite the great similarity in n-A curves between proteins, Sensitivity to surface coagulation may be an important property in biological processes and, for this reason, a closer study of the phenomenon by the monolayer technique seems warranted, One interesting observation which illustrates this is that, in solution, fully oxygenated sickle-cell hemoglobin (HbS) is much more susceptible to precipitation by shaking than normal hemoglobin (HbA), despite the otherwise great similarity in structure and properties (ref. 39,40). Other studies have focussed on the surface deactivation of cellulase by shaking and its prevention by surfactants (ref. 41) and the denaturation of e-lactoglobulinand its subsequent renaturation by dissolution in dilute acid (ref. 42).

355 OTHER TECHNIQUES FOR STUDYING PROTEIN MONOLAYERS

V.

A. Surface potentials The change in electrical potential between two electrodes (one in the aqueous subsolution and the other in the air above the surface) when a monolayer is spread is called the surface potential (AV) of the film. Two methods of measurement are in common use: 1) the ionizing electrode; and 2) the vibrating plate (ref. 43). Surface potential measurements on protein monolayers can give information on the homogeneity of the film and the orientation of dipoles in the film.

In principle,

they can also be used in conjunction with surface pressures to follow the kinetics of reactions occurring in the film.

The influence

of pH on the AV-A curves of proteins is very marked. Near the

isoelectric point and at high areas, the surface potential fluctuates wildly as the electrode is moved across the surface (ref. 44,45), showing the film to be heterogeneous. Because of the low net charge on molecules, intermolecularattractive forces (van der Waals forces, hydrogen bonding) operate to cause the formation of discrete clusters or "islands", separated by regions of very dilute gaseous film. On compression to lower areas, the surface potential becomes uniform over the whole surface, showing the film to be homogeneous (ref. 45).

Sim-

ilarly, as the pH is moved away from the isoelectric point, the region of instability of AV disappears even at the highest areas. An equation that has been used to assist in the interpretationof surface potentials is:

AV

q

4Tnu

(7)

,

where n is the number of dipoles (usually molecules) per unit area and u is the effective surface dipole moment in the perpendicular direction. The term p may in turn be interpreted by: r!=:cose

,

(8)

where E is an intrinsic moment making an angle e with the vertical. Eq. 7 has been derived by analogy with an equation attributed to Helmholtz for the potential difference between the plates of a parallel-plate condenser, assuming a surface film to consist of a planar array of dipoles. The limitations of this analogy have been stressed (ref. 46); for example, it is assumed that the dielectric constant is unity and the contribution to AV of reorientation of subphase molecules is ignored. However, the equation has been found to be useful. Frequently, the term AAV (which is proportional to p) is plotted as a function of A. By studying the variation of AV with pH for protein monolayers, it has been established that it is the contribution of the ionogenic side chains which is pH dependent (ref. 47).

Fig. 4 shows a comparison between the AV-pH curve at

356

2

6

4

a

PH Fig. 4. II-pH and AV-pH curves for insulin films on subsolutions of ionic strength; r/2= 0.01, together with the electrometric titration curve (ref. 45).

an area of 2 m2mgs1

for an insulin monolayer

0.01 and the electrometric*curve Epstein

(ref. 48).

on a substrate

for this protein

The similarity

for different

groups

is apparent

face pressure

at the isoelectric

is striking

in the AV-pH

measured

of ionic strength by Tanford

and

and the pH range of ionization curve. The minimum

point of the protein

in the sur-

is also illustrated

in

Fig. 4.

In general, AV and AAV increase with decrease of area as a protein monolayer is compressed. being oriented

Increase of AAV suggests more vertically

becomes close-packed tends to plateau pression

and segments

More detailed

compressed

films.

B. Surface

rheology

the manner

Two types of surface

studies

shear and dilational.

whereas

bulk or expansion

viscosity

high surface

dilational

by the flow through

are

AV com-

potential

changes

should prove help-

changes

occur

in these

for protein monolayers,

shear viscosity interfacial

of three-dimensional

(ns) has been measured

pressures

have been measured

Two-dimensional

three dimensions,

on further

of how the surface

in which configurational

viscosity

side-chains)

once the monolayer

begin to be pushed out of the surface,

films are held at constant

ful in interpreting

cosity

(ionized

However,

(ref. 45,49) and AAV falls off dramatically

(ref. 50).

as protein

that dipoles

on compression.

is analogous

viscosity fluids.

to that for

corresponds

to the

Shear surface

a canal under a surface

ViS-

357 pressure

qradient

(ref. 51) or by the torque on a ring or cylinder

(ref. 52) or oscillating

(ref. 53) in the surface.

fined in terms of the differences similarly

it is not strictly

cules from that of substrate protein which

studied,

viscosity

rate).

viscosity

is treated

energy

barrier.

where

The expression

to another

obtained

of the non-

Joly applied

of flow units

the theory

In this theory,

(normally

over an intermediate

for the coefficient

above

on shear

mole-

activation

of surface

viscosity

is:

k exp

(AG+ WA) kT

h = Planck's

is associated

(91

' constant

with:

and AG is

molecule

the activation

1) the work required

ently large for the molecule

done against

to form a hole in the surface

Joly concluded pearance

pressure

their size practically calculation

behavior

independent

of the activation

the area of the elementary based on the Moore-Eyring

of ns for all proteins

are summarized

Joly determined

flow units.

calculated dently,

independently

the molecules

in which

As a result,

(ref. 53) that keto imido hydrogen Thus, polyproline

gave no measureable

appear

ns, while

bonding

for other

contributed

These

and the value

in solutions

of molecular Evidence

largely

Evi-

so that segments

This is similar

no keto imido hydrogen polyaminoacids

acid

good agreement.

to diffuse

independent

65 and 69 kJ mole-‘.

in which

120 i2)

flexible

move as units.

AG and ns are approximately

Values of AG (Table 3) fell between

viscosity,

for AA (loo-

are sufficiently

long chain hydrocarbons

but still values of AA,

of plots of log ns vs. n,

The values

(on the average)

approach

and one polyamino

by Joly (90 fi2) are in remarkably

of 6-8 amino acid residues the manner

respectively,

in the monolayer

From a direct

(ref. 53) calculated proteins

and

a value of about 90 i2 for

Using a different

theory, MacRitchie

in Table 3.

at the point of ap-

of the nature of the protein.

the area of the flow unit and AG for several

results

to break all bonds

work that has to be

that all the flow units were submolecular

energy,

from the slopes and intercepts,

suffici-

to move the

to create a hole.

from the constancy

of non-Newtonian

the work required

naA is the additional

molecules.

the surface

free energy at zero II. AG

to move into; and 2) the work required

into the hole, this term including

formed with neighboring

56).

pressure,

(ref. 55) to the data.

as a movement

position

surface and

of film mole-

depended

ns at the point of appearance

of Moore and Eyring

from one equilibrium

=

surface

viscosity

was of the same order of magnitude.

the flow of a monolayer

%

and different

(i.e., calculated

for all proteins,

cules)

(II,)

the contribution

de-

(ref. 54) showed that for each

Joly

there was a well defined

viscosity

for surface

the clean and film-covered

to separate

molecules.

was non-Newtonian

However,

Newtonian

between

possible

rotating

As with IIand AV, qs is

to (ref.

weight.

was obtained

to the surface bonding

and proteins

is present

ns decreased

358 TABLE 3 Calculated values of AA and AG for proteins and one polyamino acid (ref. 53) Protein

AA

(nm*)

(kJ’Zole-1)

Polyalanine y-Globulin Pepsin

1.5 160 34

1.05 1.10 1.20

8; 67

BSA Lysozyme

70 15

1.00 1.15

:;

dramatically with increasing electrical charge. Assuming a flow unit of seven amino acid residues, the maximum number of hydrogen bonds which could contribute to AG would be seven, remembering that each residue forms two bonds but that each bond is shared by two units. A hydrogen bond energy of just over 9 kJ mole-' would then account for most of the measured surface viscosity. Measurements of nd are normally obtained from methods based on either continuous (ref. 57) or periodical (ref. 58) compression or expansion of the monolayer covered surface. In recent times, more attention has been given to dilational than to shear surface viscosity of protein monolayers. This is justified because interfaces are more often subjected to dilational rather than shear stresses. Interfacial dilational measurements would thus be expected to be relevant to emulsion and foam stability and to biological membrane systems. Pertinent studies are those of the apoprotein of the Folch-Lees proteolipid (FPi) from myelin (ref. 59) and of mixed tubulin&lipid monolayers (ref. 60).

Protein monolayers exhibit

viscoelastic rather than pure viscous behavior. For FPi monolayers, nd increased with IIin the range 37- 50 surface poise. The results indicated that,if during compression of a mixed monolayer of FPi with dipalmitoyl lecithin, energy dilssipationoccurs, it may be assigned to the protein constituent. This may be the situation in biological membranes and the alveolar lung surfacant. It was suggested that the energy dissipation results from changes in conformation of the FPi molecules (such as formation of loops and tails). In view of the previous discussion (Part IV-C),

this appears to be a realistic conclusion.

For the peripheral membrane protein, tubulin (the subunit protein of cytoplasmic microtubules), the relaxation time calculated from dynamic measurements was about five times lower than that of other protein films. The suggestion was made that this may be related to the role of microtubules in conferring mechanical stability to membranes and other cellular structures. C. Transfer of film In certain cases, it is advantageous to remove protein films after compression for more convenient examination elsewhere, For example, this method has been

359

used to study the concentration of radioiodinated BSA at the surface (ref. 61) after removal on glass slides. If a clean glass slide is lowered through a surface containing a compressed protein monolayer, no change of surface pressure is observed. On raising the wetted slide, a decrease of IIoccurs as monolayer is transferred to the slide. If the pressure is maintained constant, comparison of the decrease in area of the monolayer with the surface area of the slide shows that the film is transferred quantitatively (i.e., a transfer ratio of unity). For radiolabelled proteins, concentrations can then be determined by direct measurements in a scintillation counter. One precaution to be observed is to allow for any decrease in radioactivity,greater than the normal decay, caused by the surface (see Part VI-B). A number of different techniques have been used to study the conformation of protein and polypeptide monolayers after removal from the surface, either as collapsed or uncollapsed films. Loeb and Baier (ref. 62) deposited polymethyl glutamate (PMG) monolayers at constant pressure directly onto germanium prisms suitable for multiple internal reflection spectroscopy in the infrared. Spectra characteristicof both the a-helical and B conformdrtions were found; they were independent of the degree of composition but depended on the spreading solvent. Films spread from chloroform-pyridinesolutions containing at least 60% chloroform and collapsed onto AgCl plates for study by direct transmission IR spec-1 amide 1 and 11 absorption peaks at 1654 cm-l and 1550 cm ,

troscopy showed the

respectively,which is characteristic of both the a-helical and random coil conformations. As the pyridine concentration in the spreading solvent was increased,the monolayer spectrum showed increases in intensity of bands at 1625 -1 -1 -1 cm and 1520 cm at the expense of the 1654 cm and 1550 cm-l bands, respectively. Amide 1 and 11 absorptions at 1625 cm-' and 1520 cm'l bands are associated with the extended chain 6 configuration of polypeptides. The behavior is illustrated in Fig. 5

for films that had been removed after compression beyond

the monolayer collapse point. Similar results were obtained for this polypeptide by Goupil and Goodrich (ref. 63) who also used a suitable choice of spreading solvent to vary the conformation and infrared spectra of films transferred OntO

germanium prisms by the Langmuir-Blodgettmethod, They also showed how sur-

face viscosity measurements could be used to differentiate between the a and 8 conformations. It was postulated that a surface film of folded, independent rods (a-helix structure) should be less viscous than a coherent interlocked

chains

(B-structure).

set of unfolded,

This appeared to be the case,

The tempera-

ture dependence of the surface viscosity was different for the two conformations. The B conformation was characterized by a high surface viscosity which fell with increasing temperature, possibly due to weakening of the intermolecularhydrogen bonding. On the other hand, the surface viscosity of the polypeptide in the u

360

Wavenumber

cd

Fig. 5. Infrared spectra of collapsed films of PMG spread from _. spreading solutions of varying chloroform/pyridine content. Numbers on the figure refer to percent chloroform in the spreading solution (ref. 62).

form showed a relatively

low surface

increasing

This was thought

temperature.

viscosity

which

increased

irreversibly

to be due to conversion

of the

with

CL to

the B structure. An extensive

study of the conformation

moval of compressed

surface

By means of infrared

of synthetic

films has been carried

spectroscopy,

polypeptides

out by Malcolm

it was shown that material

face films of a range of polypeptides

was in the a-helical

addition

introduced

method

to infrared

spectra,

for monitoring

of requiring information. men is low.

Malcolm

the conformation.

very small amounts Infrared

spectra

Electron

of material

electron

after re(ref. 64,65).

removed

diffraction

diffraction

The study of transferred

In

as another

has the advantage

yet giving detailed

conformational

are more useful when the crystallinity

bility exists that conformational

from sur-

conformation.

of a speci-

films is an indirect method and the possi-

changes

may occur on removing

the film from the

surface,

In order to check this point, Malcolm developed a method for studying

deuterium

exchange

in polypeptide

monolayers

peptides were spread at the surface sion, dried and the infrared mined

from the relative

spectra measured.

strengths

(ref. 65).

for measured

times,

The N-deuterated

poly-

removed after compres-

The extent of exchange was deter-1 of the NH and ND stretching bands at 3300 cm

361 and 2400 cm where

-1

confirmatory

Rates of exchange

, respectively.

hydrogen

bonding

evidence

for the a-helix where

bonded and would not be expected Most of the conformational

form, proteins

only partially

studies

red spectra of paramyosin

polypeptide

amounts

of the molecule

technique

known, whereas

as a rough

index of conformation.

found for transferred compared

after spreading

proporthe infra-

contains

and

is one of

appreciable

The ratio of absorb-

to that at 1655 m-l (a and random) was taken Values

for this index of 0.6 and 0.9 were

monolayer-s of paramyosin

and s-lactoglobulin, solution

results were taken to mean that the B structure proteins

in

as monolayers

Paramyosin

at 10 mNm-'.

s-lactoglobulin

to 1.1 for residue from the spreading

by spreading

pro-

have their chains

larger or smaller

in the B and random chain forms.

ance at 1635 cm -' (B configuration)

on model

Loeb (ref. 67) has measured

and B-lactoglobulin

proteins

intramolecularly

chains may exist exclusively state generally

form, with relatively

by the Langmuir-Blodgett

the most helical

groups are

with water molecules.

in their solution

in the a-helix

this was taken as

to date have concentrated

tions in the 6 form or random structure.

transfer

peptide

to exchange

Whereas

teins, the polypeptides. the a-helix

were much slower than in polymers

to water can take place; therefore,

respectively, The

of s-lactoglobulin.

does not appear

at the surface and that the a-helical

to be induced

structure

is main-

tained as found for the polypeptides. Optical

rotary dispersion

(ORD) and circular

to study the confortmttion of polypeptides CD has been applied

to the study of corresponding

plates by the Langmuir-Blodgett aligned

identically

L-alanine

the film balance.

the orientation

True OD spectra, at numerous

The results were consistent in the a-helical of the incident

light.

copy after transfer

monolayers

(ref. 68,69).

exhibited

deposited

and unlabelled

expected

has been examined to quartz

plates

and B-sheet

molar absorptivity

protein.

a planar sample

explained

or changes

effects

light beam.

of polypeptides to the direction

by UV and CD spectros(ref. 69).

CD spectra

in the film oompared

of 11,950

It was proposed

of

were obtained

1 mole cm"

to values of 9,100 and 9,600 for isotropic

from the effect of orienting by solvent

properties

with the helix axis perpendicular

amount of a-helix

194 nm was found compared

which could

chains on the surface

about the axis of the incident

B-lactoglobulin

on quartz

films of poly-

free of the linear component,

with the optical

An enhanced

Recently,

When all plates were

linear dichroism

of the polymer

from a film balance

an increased

of labelled

angles

conformation

that in solution,

(CD) have been used

in solution.

in the light beam of the spectropolarimeter,

be used to determine

suggested

method

and poly-y-methyl-l-glutamate

on films oriented

dichroism

and proteins

to

at 193-

solutions

that this results

mainly

in the light beam and could not be

in conformation

of the protein.

Measurement of contact angles after transfer of protein monolayers to Langmuir-Blodgettslides is another technique which yields useful information on the orientation of polar and non-polar groups (see Part IV-D). D. Direct optical methods Spectroscopy has been a powerful tool for studying molecular structure in bulk solution. Because of the small amount of material present in a monolayer, some method must be used to amplify the spectral signal. One approach is to use parallel mirrors to pass light through the film many times. An example of this technique applied to protein monolayers is the interesting study of films of oxyhemoglobinsA and S at the air/water interface (ref. 70). As mentioned (Part IV-D), these hemoglobin variants display different surface behavior as evidenced by their tendencies for mechanical precipitation. Absorption maxima were shifted from the solution value of 415 nm to 419 nm for both HbA and Hbs, consistent with a conformationalchange in the direction of greater unfolding, although shifts were the same for each. These Soret band maxima did not shift on compression of the films. It was noted, however, that surface isotherms were more expanded for HbS than HbA, indicating a greater unfolding. Ellipsometry is a sensitive method for measuring the thickness and refractive index of thin films at surfaces. The technique has been mainly used for studies of films adsorbed from solution (ref. 71,72) but can be applied equally well to monolayers. De Feijter et al. (ref. 73) compared measurements of surface concentrationsof protein monolayers by ellipsometry with results for two other independent techniques: 1) a spreading technique by which known amounts of protein are spread on a known area of surface; and 2) a radiotracer method using radioactively labelled protein. For B-lactoglobulin,two different spread-2 calculated from the amount ing experiments gave values of 0.98 and 1.09 mgm -2 spread and 1.00 and 1.05 mgm calculated from ellipsometer measurements. For -2 -2 by the radiotracer a-casein, ellipsometry gave 2.5 mgm compared to 2.7 mgm method. From these figures, the reliability of ellipsometry is evident.

In view of the inherent uncertainty in drawing conclusions from measurements on films transferred from monolayers (particularlyin relation to conformation determinations),there is a need to develop further the direct methods for examining protein films _in situ. These methods have the great advantage that the films can be manipulated and measurements made as a function of different variables such as surface pressure and time. Techniques such as those developed by M6bius and co-workers, including enhanced light reflection (ref. 74) for direct measurements as well as quenching of fluorescence (ref. 75) for transferred monolayers may prove valuable for studying mixed monolayers and two-dimensionalreactions of proteins.

363 VI.

DESORPTION The question

FROM MONOLAYERS

of whether

a controversial

adsorption

that it is an irreversible monolayers,

despite

and Schaeffer

process

of a monolayer The equation

= akT

increase

per cm* at the surface

with the film. 2 -1 on the surface 1 m mg

and c is the concen-

According

There-

10s8.

to this, an increase of film pressure

between

of compressed

and has a molecular

o would have a value of 2.85x

of the film by a factor of 10 g5. The

the solubility

lack of correspondence

the great stability an argument

to the

in equilibrium

fore, at 20°C, dln c= 220 dn. of 15 mNm -' should

pressure

(11)

(e.g., ovalbumin),

of 35,000

from

Langmuir

solution.

theory of the effect of surface

,

in solution

apparent

held belief proteins

(ref. 77) (based on the Gibbs equation)

For a protein which occupies weight

of desorbing

used was:

where u is the number of molecules tration

or not has been

a widely

of many in aqueous

a general

case of proteins.

is reversible

that has prompted

is the difficulty

the high solubility

(ref. 76) applied

on the solubility

dn/dlnc

of proteins

One observation

topic.

the predicted

solubility

protein monolayers

in support of the irreversible

increase

has been considered

nature of protein

and to be

adsorption.

A. Kinetic measurements Langmuir of proteins

and Waugh

and their enzyme

sure displacement

pressure

expulsion solubility

effects of increasing

of portions involves

displacement

of molecules

desorption

on monolayers

They distinguished

between

pres-

is equivalent

in Part IV-C,

discussed

of complete

times in solution

of monolayers

of compression

Pressure

solubility.

digestion

sures on the solubility

the effects

(pepsin) digests.

and pressure

to the reversible whereas

(ref. 78) studied

molecules.

and increasing

of insulin and its digests

The

surface

pres-

are summarized

in Table 4. With increasing

digestion

These are the figures reference

pressure

time, losses of material

in the first row at n=mNm-',

for measuring

refer to the loss of monolayer originally

For ovalbumin,

relatively

initial molecular A preliminary

as a percentage

lower proportions

conditions

Other figures

of the total material

to compression

for 10 min. periods.

of pressure-soluble

than for insulin,

theory

consistent

material

were

with its higher

to explain

This theory

pressure predicted

solubility

was proposed

that polypeptides

If pressure solubility

is observed

between

by Langmuir

should show pres-

in the range II= 5 to 10 mNm-' when the molecular

1200.

increased.

weight.

(ref. 78).

sure solubility proximately

expressed

spreading

losses of monolayer.

spread when films were subjected

found under comparable

and Waugh

permanent

during

which was taken as the

weight

is ap-

II= 20 and 25 mNm-',

364 TABLE 4 Losses of monolayer for insulin and its peptic digests during spreading and 10 min. compressions at different surface pressures. The first row (1 mNm-I) gives the amount of protein (as a percentage of total spread) lost during spreading. The last row (residue)gives the amount of monolayer (as a percentage of total spread) remaining after spreading and compression at each of the six pressures (ref. 78). --."-1 (mNm )

Time of digestion 21

0

: 10 15

!*': 5:2 3.0 87.0 --

a molecular

weight of ca. 1700 is indicated.

tion products

of insulin

had molecular

The first quantitative

measurements

tein monolayers

were reported

Because of the superimposed pressure sured

displacement

a given pressure. face pressure

relaxation

by Langmuir

higher surface 79) therefore of area. constant

However,

process described

and Waugh),

desorption

(15 mNm-' and above). pressure

for a measured

The permanent

(called

be simply mea-

of surface

recoverable

area at

at low sur-

losses are only observed Gonzalez

(A,),

and MacRitchie

at

(ref.

permanent

losses

After maintaining

time, the surface was completely

(area A).

in Part IV-C cannot

of 5 mNm-I to monitor

at 5 mNm-I

from pro-

(ref. 79) for BSA.

by the decrease

losses of area are totally

The area was first measured

that the degrada-

in the range l,OOO- 2,000.

and MacRitchie

substances)

chose a reference

pressed to 5 mNm-'

::: 4.5 3.0 1.7

of the kinetics of desorption

(10 mNm-I and below) and permanent pressures

70 4.5

It was concluded

weights

by Gonzalez

(as with other monolayer

52

46 1.4 4.0 10.9 7.6 4.3 25.8

i.7

22: residue

in hours

expanded

n

and recom-

loss of area was then given by

A,-A. Two checks were used to determine due to desorption.

tion of time at a fixed pressure BSA in the subphase.

whether

the permanent

concentration

the rate (in the presence system continued

the idea that desorption

Initially,

gentle

of

the kinetics of

stirring

the rateand with

the rates were the same but after a time,

of stirring) to decrease

increased,

The second check was to compare

time curves at this same value of IIwith and without no BSA in the subphase.

as a func-

of 25.6 mNm-' with varying concentrations

As the subphase

area loss slowed as shown in Fig. 6.

stirred

area losses were

In the first, rates of area loss were measured

became constant

while that in the un-

with time (Fig. 6).

was the cause of the permanent

The results area losses.

support

365

P

I

1

40

80 (min)

Time Fi

. 6. Rate of desorption-time curves for BSA monolayers at 25.6 mNm-I pressure. 3 : no protein in subphase no stirring; (o ): stirring; (A): 0.008% BSA in

!.:bphase; (A):

In desorption

0.05% BSA in'subphase;

from monolayers,

between the monolayer

an equilibrium

and an adjacent

In the absence of convection layer

(0): 0.10% BSA in subphase

(which is maintained

is usually

thin subsurface

and back-diffusion,

at constant

t, is then given by the corresponding

established

diffuse

from this

to the bulk phase,

n, of molecules

equation

rapidly

layer of bulk solution.

molecules

concentration)

The number,

ially at zero concentration,

(ref. 79).

that desorb

from classical

init-

in a time,

diffusion

theory

(ref. 80):

n = 2c,

IT I i DJ

l/2

iw

,

where co is the subsurface is 3.14.

concentration,

The rate of desorption

D is the diffusion

at any time, t, follows

coefficient

and

II

of

by differentiation

Eq. 12:

t-1/2 Gonzalez several

(13)

and MacRitchie surface

did not extrapolate diffusion

(ref. 79) plotted 2

pressures. to zero.

This is consistent

but with an energy barrier

the slopes,

vs. t-1'2 for BSA monolayers

The plots were linear although

with a process

to the desorption

values of co (the steady state sublayer

lated and are shown in Table 5, together

at

lines of best fit controlled

step (ref. 81).

concentration)

by

From

were calcu-

with values of co, at corresponding

366 TABLE 5 Calculated

subsurface

concentrations

n

of BSA (ref. 79) C

cO

(from &d;;ption

(mNm-l)

(from adiorption isotherm)

(g cms3)

(gem -3 1

28.8

1.28 x 1O-6

5.5 x 10-2

27.2

1.94 x 1o-6

2.2 x lo-2

25.6

4.86 x lO-7

9.1 x 10-3

24.0

2.62 x lO-7

3.8 x lO-3

22.4

5.7 x 10-8

1.6 x lO-3

pressures,

from the equilibrium

ancy between

adsorption

isotherm of BSA.

the two sets of values for co is confirmation

The large discrepfor the existence

of an energy barrier at the surface. Returning protein

to the results

in the subphase,

and the desorption interface, diffusion

shown in Fig. 6, it can be appreciated

the concentration

rate would be expected

there is a stationary only.

tionary

to decrease,

conditions.

layer, where material

we can calculate

in thickness.depending

Near every occurs

by

on the type of inter-

For the results of Fig. 6, the introduction

gradient

is carried

the thickness

is lowered

as observed.

causes the rate of area loss to become constant

At this point, the concentration

that with

near the surface

layer through which mass transport

This layer varies

face and the stirring of stirring

gradient

has extended

after about 30 min.

to the edge of the sta-

away by convection.

of the diffusion

layer

For this example,

(6) from the equation:

(14) Using values of 6.0 x 10e7 cm2 set -' for D, the diffusion coefficient of BSA -7 -3 (ref. 82), 4.86 x 10 for co (from Table 5) and zero for c, the effective g cm concentration

at the edge of the diffusion

lated for 6.

Values for the width of the stationary

have been found to vary between about 0.03

mm where

stirred

vigorous

subphase

We may utilize -1 to estimate mNm the equation:

stirring

thus appears the results

layer, a value of 0.08 mm

1.0 mm for unstirred

is used (ref. 84).

is calcu-

layer in aqueous systems

solutions

(ref. 83) and

The value of 0.08 mm for a

to be of the right order. for the steady state desorption

the magnitude

of the energy barrier

of BSA at 25.6

to desorption

by applying

367 dn _ 'o --R1+R2 dt where

(15)

’

RI is the diffusional

resistance

and R2 is the interfacial

is equal to i and thus has a value of 1.3x lo4 set cm for g

and co gives a value for R2 of 2.4x lo* set cm

the kinetics

of desorption

in controlled probable

mainly

to a cooperative

must leave the interface

B. Radiolabelled

protein

bility of monolayers

as it provides

losses of compressed

monolayers

the absolute

rate

It appears

resistance.

step in which all segments

of a

a reliable

method

for studying

way of determining

configurational and labelled

changes

the sta-

if permanent from the surface

or coagulation.

with 1251 was carried

A out by

(ref. 8). Permanent losses of area were -1 of 18 mNn and above. Monolayers were compressed

and maintained

then expanded

and recompressed

the permanent

loss of area before

samples

compression

was measured

at constant

to a reference

of the monolayer

and radioactivity

repeating

pressure pressure

in r~ corresponding

together with the measured remained

successive

For example,

for 20 min. periods, -1 to determine

of 10 mNm

the cycle.

For the radiolabelled

were removed on glass slides

in a scintillatian

counter.

cycles are shown in Fig. 7 for radiolabelled

Radioactivity following

although

simultaneously.

is a valuable

of BSA unlabelled

at pressures

to a given pressure

decreases

Therefore,

RI

of values

and Ter-Minassian-Saraga

only observed

protein,

.

are due to removal of molecules

such as molecular

study of monolayers MacRitchie

Substitution

proteins

The use of radiolabelled

or to processes

-1

resistance.

.

of diffusion,

by the much larger interfacial

that R2 is related

given molecule

show the influence

-1

(see Part V-C)

Examples of the

BSA at 21 mNm-'.

The

to the removal of slides at 10 mNm-' are shown -1 -2 counts in min cm for the transferred films.

constant

compression

within

experimental

cycles,

error at all pressures

as also did film compressibility,

after

losing 68.6% of the film at 27 mNm-I, the specific activity -1 -2 counts min cm compared to an average value of 3620 counts

was 3720+400 -1 $2 min for measurements gimes) at 10 mNm-'.

on 18 slides of BSA films

This is consistent

the remaining

monolayer

from analyses

of radioactivity

being unchanged.

(removed after varying

re-

with an area loss due to desorption, Confirmation

in the subsolution

of desorption

was obtained

after a known loss of material

from the surface. From results

such as those in Fig. 7, it is possible

to separate

sible and irreversible

area losses at a given pressure.

tracting

loss (from the area change at 10 mNm-')

the permanent

loss (measured at the high pressure over the 20 min. period). shown in Table 6.

the rever-

This is done by subfrom the total Some values are

368

125 Fig. 7. Compression-expansion (not shown) cycles for I-labelled BSA monolayer held for 20 min. periods at 21 mNm-I. A is in arbitrary units. Surface area may be calculated from the fo mula: area= 8.33At10.7 cm2. Amount of protein spread= 80 1 of a 0.62 mgml -1 solution. The numbers correspond to the measured counts min- y cm-2 for the films transferred onto the glass slides (ref. 8).

TABLE 6 Reversible changes tervals at various

and irreversible losses of BSA monolayers surface pressures (ref. 8) Decrease

II

(mNm

-1

of film area

Decrease (total)

during

20 min. in-

(% of total) Change++ (reversible)

Loss+

:Y

22 51.5

:

44.5 21

z': 30

80.4 71.5 82.2

9 15 18

62.5 65 65

'Determined

tt

Difference

One

at n= 10 mNm-' between

total decrease

and loss (columns 2 and 3)

factor that needs to be allowed

proteins

is the possible

relatively

for in surface

rapid decrease

natural decay rate) when they remain at the surface of about 2% per hour was reported face catalyses

a reaction

stancy of specific losses

implied

in which

activity

in this work. iodine

using

in radioactivity

It was suggested

BSA film following

(calculated

I-labelled (above the

for long periods.

A decrease

that the sur-

is lost from the surface.

in the labelled

that the tagged molecules

studies

The con-

irreversible

to be only one in 90 of

area

369 the total) desorbed

at the same rate as the untagged

found that the sample of labelled above pressures altered

of 20 mNm

the protein

C. Desorption

in biological

monolayer.

sample had

y-globulin

and catalase

A striking

the mixed

films are initially

The area coverage

of insulin

The rate then deviates

and catalase.

is one

identical

from the surface,

(T-globulin

studied

is that the rates from

was

to 25- 30%.

film, the area approaching

value, the desorbable

(min

after spreading

is reduced

or catalase).

leaving a monolayer

Time

of 17.5 mNm-I,

to those from a pure insulin monolayer.

until the coverage

at this moderate

and

is il-

loss of area over long

behavior

in the mixed films immediately

protein

for from

B-lactoglobulin The behavior

At the pressures

show no permanent

from that of the pure insulin

that of the non-desorbable

tively removed

(ref. 85).

of insulin at a pressure

feature of the desorption

50% and the rate is constant

ing the film pressure

of two proteins,

has been studied

with y-globulin

periods.

processes

(lipids and other proteins)

useful to know how a pure protein desorbs

in Fig. 8 for the desorption

alone and in admixture

about

treatment

systems and industrial

The desorption

from mixed monolayers

(~20 mNm-I),

faster than the unlabelled

that the labelling

compete with other compounds

It is therefore

a multicomponent

lustrated

BSA desorbed

This suggests

it was

in some way.

situation

proteins

the interface.

insulin,

.

However,

from mixed monolayers

The practical in which

-1

ones.

Thus, by maintaininsulin

is effec-

of the pure component

that

1

Fig. 8. Log A-time plots for desorption of insulin from its pure monolayer (0); from a mixed monolayer with T-globulin (0); and from a mixed monolayer with catalase (0) at 17.5 mNm-I on pH 7.3 buffer. Dashed line corresponds to area of pure v-globulin and catalase monolayers (ref. 85).

370 is non-desorbable

at that pressure.

for mixed monolayers 86).

It evidently

the sublayer systems

arises

This behavior

desorbable

because

to date,is

layer providing

the concentration

practically

its surface coverage

low this coverage,

is greater

to maintain

at different

surface

for mixed

films are relevant

occurring

in blood clotting.

cesses across

interfaces

in

and, for the

from the monolayer concentration.

are summarized

to competitive

protein

pressure

alone and in admixture

pressures

(ref.

of the mono-

than 25- 30% of the total.

the sublayer

adsorption

Some results

in systems

The results

such as those

for transport

where multicomponent

Be-

to the

with non-desorbable

in Table 7.

They also have implications

or membranes

compounds

of the composition

the rate of supply of molecules

is insufficient

to be quite general

of desorbable

by the surface

independent

for films of insulin and B-lactoglobulin components

appears

and non-desorbable

(co in Eq. 12) is determined

studied

sublayer

containing

pro-

films are present.

TABLE 7 Rates of desorption of insulin and B-lactoglobulin surface pressures on pH 7.3 buffer (ref. 85). Monolayer

from monolayers

Rate of desorption 15+

Pure insulin Insulin - DPPC Insulin - y-globulin Insulin - catalase

17.5+

zo+

pressure

The displacement

and catalase

trolling

desorption;

Whereas

(Fig. 8), provides namely,

insulin

Z

at surface

size.

The same is suggested

of several

desorption

products.

proteins

by the MacRitchie

spanning

a range

where the rates could be conveniently

in Table 8.

it can be seen that as the molecular

the rate of desorption

y-

to one of the factors con-

and their degradation

pressures

Results are summarized

by the large proteins,

a pointer

the molecular

the rates of desorption

weights

In general, creases,

14 15

weight

and Waugh on proteins

(ref. 85) studied

measured.

11 12

30+

:; 60 61

22: 26

of the small protein

globulin

of molecular

27.5+

(mNm-')

D. Effect of molecular

work of Langmuir

(min-l x 103) 25+

24 ; 6 6

Pure 6-lactoglobulin s-lactoglobulin - DPPC +Surface

at different

at a given surface

weight of the protein pressure

decreases

in-

markedly.

(molecular weight 6,000) is large enough to be -1 measured at a pressure of 15 mNm , the larger-sized proteins such as y-globulin -1 and catalase are stable at this pressure and require pressures above 30 mNm for comparable

of insulin

desorption

rates.

In most studies of protein monolayers,

it is

373 TABLE 8 Rates of desorption

of five proteins

Mol. wt (x 10-3) Insulin B-Lactoglobulin Myoglobin y-Globulin Catalase

unusual belief

6 17.5

15+

ZO+

56

530 20

25+

30+

50 34

90 67 9

:;0 230

pressure

pressures

(ref. 85).

35+

40+

45'

144 20 30

40 70

110

(mNm-')

for this higher range of pressure that protein

stability

surface

Rate of desorption -1 (min x lo4

Protein

'Surface

at different

adsorption

at lower pressures)

to be investigated.

is irreversible is therefore

The widely

(based on observations

understandable,

although

held

of their unjustifiable.

E. Theory of desorption In any explanation

for the observed

desorption

behavior

of proteins,

the

following results need to be considered, Most proteins are stable up to 15 -1 mNm and desorption is not usually detectable below that surface pressure; this despite ficient tion.

the fact that equilibrium

equilibrium Above

concentrations

15 mNm_l,

than would be expected trations,

indicating

stantial magnitude. as molecular

weight

adsorption

desorption

can be measured

from calculations

the existence

would

predict

to cause measurable

suf-

desorp-

but rates are appreciably

using the equilibrium

of an energy

From a comparison increases,

isotherms

in the sublayer

sublayer

less

concen-

barrier at the surface of sub-

of different

proteins,

the rate of desorption

it appears

that,

at a given surface

pres-

sure decreases. An attempt

to quantify

been made by MacRitchie

the free energy barrier

(ref. 34).

step is taken to be a molecule of ca. 1 nm2.

process,

The transition

whose trains

This is the calculated

for a protein molecule

to adsorb.

when a protein molecule

equilibrium

and there may attain

It is therefore is compressed

trains,

is a finite probability the transition

configuration fluctuation

between

energy

required

assumed

occupy an area to be cleared

that in the reverse

to this area,

it is no longer

pressure,

state.

fluctuations,

At low pressures,

there is a (see Part IV-C)

individual because

molecules

the average'

in the form of trains,

be prohibitively

has

in the desorption

segments)

At any surface

that through

for this would

of proteins

loops and tails of a molecule

is one with nearly all segments required

complex

(adsorbed

area of surface

stable at the surface and will desorb. dynamic

to desorption

the energy

high and the probability

372 almost zero.

However,

the form of trains A quantitative

estimate

given surface

pressure

tein (see Fig. 3). required

to compress

(nc) at which

I

pressures

Adn

of the free energy barrier

to desorption

the molecule

required

(aGdes) at a

II-A curve of the pro-

to remove a molecule

from the given pressure

it occupies

in

increases.

an area of ca. 1 nm*.

will be that

(II,) to the critical This is given by:

.

(16)

e

The integral appropriate

can be evaluated

from the area under the n-A curve,

limits of Il. Because

of the sigmoidal

this area (and thus the free energy) rapidly

as Re increases

different

surface

constants

for desorption,

are compared desorption decreases

of such a fluctuation

C

AGdes = n

creases

where the proportion of segments

may be made from the equilibrium

The free energy

pressure

II

at surface

is much lower, the probability

pressures

15 mNm

-1 .

for BSA together

Some calculations

with corresponding

rapidly and attains

to the range of surface values relative

of AGdes at

measured

and MacRitchie

It can be seen that the appearance

rates corresponds

the

is very large at low values of ne but de-

above

from results of Gonzalez

in Table 9.

between

nature of the n-A curve,

rate

(ref. 79),

of measurable

pressures

at which ~~~~~

to kT where rate processes

become

significant. This simple theory appears tion.

to explain

significant compressed

desorption.

The rapidly changing

at higher surface

fewer trains molecules

per molecule)

of different

same distribution

pressures

causes

molecular

of segments

trains and hence the correspondingly consistent

with observations

REACTIONS

Many biological

trains,

reactions

proteins

ever

If we compare two

(at a given pressure)

the

loops and tails, the large mole-

of the greater

number of segments

larger area occupied

occur at interfaces.

participate

reactions,

trough or Wilhelmy-type

and control

by them.

Proteins,

as

This is

new instruments

phenomena.

reactions

Spread monolayers

In order to study

based on modifications

have been introduced.

with lipids,

of living cells.

of interfacial

and many enzyme reactions.

for these complex

film balance

together

the functioning

in a wide variety

respiration

simple model systems

interfacial

leaving

any when

(see Table 9).

which surround

such as blood clotting, provide

of segments

and assume

preclude

of molecules

IN MONOLAYERS

form the membranes

In addition,

pressures

~~~~~ to fall rapidly.

between

of protein desorp-

configuration

(expulsion

weights

cule will have a higher ~~~~~ because

VII.

the main features

The very large values of ~~~~~ at lower surface

of the Langmuir

TABLE 9 Calculated free energies of activation for desorption for desorption of BSA monolayers at different surface

n

Rate constant for desorption

AGdes/kT

AGdes (uJ molecule-')

(mNm-I)

(Atides) and rate constants pressures (ref. 34).

(gcm-2sec-1'2x 0 20 22.4 24.0 25.6 27.2 28.8

2.6 4.3 2.6 1.7 1.0 5.8 3.6

x x x x x x x

10 10 10 10 10 10 10

660 106 t:

lOI*)

0.42 1.83 3.50 7.50 9.17

24 14 9

A. Instrumentation Troughs

composed

one subphase

to another

the instrument ments

of several

have proved

of Fromherz

Each of the axles

be coupled

assembly

is connected

composed

thus compressing

monolayer

to be transported are separated

the trough.

This arrangement

and transferred

to another

may be quenched

by moving

occurring

where

reaction

over one or more compartments

tion of the monolayer by depositing Another

by walls which are slightly allows monolayers

important

development

monolayers. subphase, action

When lipid monolayers the reaction

is followed

area decreases compartment

products

at constant

according

The slope of the plot is directly expressed

no reactants.

by standard

become soluble

if enzyme

The

surface

trough

of enzyme

by lipases

techniques

introduced

present

away.

or

analysis.

reactions

in a conventional

curve of Fig. 9.

is

The composi-

chemical

and diffuse

pressure

a reaction

where reaction

for subsequent

by a small canal

it is

pressure.

Alternatively,

the kinetics

to the left-hand

trough connected

a linear plot is obtained

reaction,

containing

are hydrolyzed

surface

surface

when

an enclosed

lower than the edges of

has been the zero-order

Verger and de Haas (ref. 89) to measure

whereas

the

to be spread on one subphase

proceeds.

may then be determined

concentric-

to the frame,

enabling

from the subphase

one or more layers on slides

In

The inner axle may

the surface,

move together,

the monolayer

pivoted

barrier.

along the trough at constant

compartments

from

(ref. 87,88,89,90).

it is coupled

If

of monolayers

trough with eight compart-

of two axles

to a movable

to the outer axle, both barriers

transfer

useful

a circular

to the outer axle or the frame.

motor moves only one barrier, coupled

to allow

particularly

(ref. 87,88),

is used with a driving

ally.

compartments

by

in

in the

If the retrough,

the

By using a two-

(as shown on the right in Fig. 9),

is present only in the left compartment.

proportional to the velocity of the enzyme -2 -1 cm min since, in this case, the

in units of molecules

total number of substrate

molecules

accessible

to the enzyme

remains

constant.

37'4

First

order

,‘I

trough

1. 4

*

I

8 Time

Zero order

a.

12 min

I,

1 .

16

I 4

trough

I

I

I

8 Time

1

I

.

12 min

I

(

16

Fig. 9. Comparison between the recorded kinetic plots obtained with a standard trough (left) and with a "zero order trough" (right) (from ref. 90).

Most of the studies by having

on enzyme

the substrate

the subphase.

reactions

at interfaces

spread as a monolayer

Nevertheless,

some experiments

have been carried out

with the enzyme

in solution

using spread monolayers

in

of enzymes

have been reported.

B. Enzyme reactions

and retention

The usual procedure enzymes

for investigating

by transfer

to solution

vantage of this approach measured

It would therefore activity devised

of enzymes a method

and assay of the activity.

configuration.

in cell membranes be valuable

for measuring

to be studied.

or attached

in situ. --

the activity

spreading

The obvious

disadbeing

are known to function

to intracellular

interfaces.

these systems by studying

For this purpose,

the effects of surface

pressure

the

Skou (ref. 91)

of acetylcholinesterase

To avoid interference

on

film on a slide,

is not necessarily

Many enzymes

to try to emulate

as monolayers

layer state, which permitted unfolding

or collapsed

is that the enzyme activity

in its interfacial

incorporated

activity

the effect of surface

has been to remove the enzyme monolayer

followed

while

of biological

in the mono-

and the degree of

caused by dissolution

of enzyme

into the subphase

during

the monolayer

spreading,

ing) to a new compartment

of a multicompartment

new compartment

the acetylcholine

and samples reaction

contained

removed

occurring

The measured

preted

higher

pressure

was a maximum

times.

favorable

pressure

conditions

the measured of enzyme

and also with the surface

before compression

on further

enzymes

for maximum

was calculated

of the protein.

the orientation

was 10 mNm-I.

was inter-

off at lower and of side-

Under the most

time before compression

to be 51% of the activity

that this approach

in the monolayer

and 10 mNm'I),

of the same amount

frequency

after removal

does not give direct

protein molecules

after unfolding

in the two cases.

Skou

of enzyme

of enzyme with

There

is thus a

of the retention

of biological

films.

this type of experiment

Although

about activity spreading

revert

at the surface.

Although

ref. 92).

for each case to allow a more valid comparison.

from surface

of whether

up with studies

that the rate of collisions

factors

information

to the question

and whether

(see, however,

may be quite different

There have been a number of studies of proteins

has not been followed

state with those for equal amounts

it should be remembered

molecules

need to calculate

relate

to reflect

activity

spread as monolayers

activities

in solution, substrate

was assumed

The time

pressure.

in solution.

It seems surprising

compared

in this was stirred

of the enzyme

of the monolayer

of the degree of unfolding

(i.e., 30 sec. elapsed

activity

the kinetics

when this time was 30 sec., falling

The pressure

Optimum

chains.

The subphase This subphase

of the enzyme was found to vary with the time allowed

by the author to be a measure

The activity

(after spread-

to be measured.

before compression

at zero surface

trough.

substrate.

for assay, allowing

at the surface

activity

after spreading allowed

at intervals

was transferred

in the surface

of proteins

to their original

activity

state, it does

is a reversible solution

process

configuration

In some of the studies, a total loss of activity

has been found following

compl.ete spreading

are removed

films or fibers, care needs to be taken in interpreting

as collapsed

results as surface Part IV-D). activity antigenic

Other studies

may be retained activity

after spreading suction proteins

coagulation

Relatively

after taking

freeze drying.

after surface

of the soluble

spreading.

surface

and removal

most of the was recovered

by compression

(lo- 29%) were reported

losses caused

(see

and serological)

of paramecium

from the surface

When enzymes

in proteins

(enzymatic

For example,

proteins

small losses

into account

(ref. 93).

large changes

have shown that biological

as monolayers

(ref. 94).

itself causes

of enzymes

by the spreading

and

for three

solvent

and

376 C. Other reactions The unfolding molecular

of protein molecules

secondary

valence

molecularly,

this being favored

Furthermore,

reactive

ientation

groups,

of molecules,

layers, provide

previously

suitable

tively few studies of reactions been to measure of dissolved

changes

substances

with those on polyamino identify

at an interface

shielded,

attained

for high reactivity.

in mono-

There have been rela-

protein monolayers.

One approach

induced

in the subphase.

the effects

By comparing

The or-

possible

in II-A or n,-A characteristics

acids with various

inter-

in monolayers.

may become exposed.

to the high concentrations

in spread

causes intra-

These bonds may re-form

by the high concentrations

in addition

conditions

on spreading

bonds to be broken:

side-chains,

has

by the presence

it becomes

of proteins possible

to

the site of reaction.

Low concentrations (gelatin,

collagen

to show considerable interaction

of silicic acid in the subphase

and human serum albumin) hysteresis

served for a poly-DL-alanine tion of hydrogen

monolayer

bonds between

acid and the carboxyl,

viscosity,

protein monolayers

Because

the vicinity

of their isoelectric This is illustrated

(ref. 95).

(SiOH) groups of polymerized

technique

Proteins

either side.

curves

The

the same effect was ob-

groups of the protein.

of the silicosis

ns, is a useful

(ref. 53).

compression

and

, interaction was interpreted as the forma-

the silanol

amino and ketoimino

of this study to an explanation Surface

in successive

took place in the pH range 2-8.

caused protein monolayers

to become much less compressible

process was discussed. for detecting

show a maximum

points,

silicic

The relevance

decreasing

interactions

in ns at pH values

in in

as the pH is moved away on

in Fig. 10 for monolayers

I

of pepsin

(I.P.-2)

I

PH (A): BSA; (A): Fig. 10. Log no vs. pH for monolayers: glutamic acid; (o ): poly-L-lysine (from ref. 53).

pepsin;

(0): poly-L-

311 and BSA (I.P.

Poly-L-lysine

-4.5).

arly show a drastic

in ns as the electrical

decrease

This effect was thought

is increased. ular keto-imido

hydrogen

set up in the surface presence

and poly-L-glutamic

bonds as a result of the electrical

chloride

Protein monolayers

of metal

solution,

simil-

charge on the molecules

to be due to the breaking

(see Part V-B).

of low concentrations

0.001 M mercuric

acid monolayers

ions in solution.

a BSA monolayer

of intermolec-

repulsive

forces

are sensitive

to the

On a subphase

showed an increase

of

in no

(the value of ns when the log ns-R is extrapolated

to II= 0) of about 60 surface

poise (typical

Polyalanine

for a number of proteins

by this subphase similar

manner

whereas

to the proteins.

with the ionizable side-chains,

carboxyl

possibly

If lipid molecules is observed, is compressed, and decreases reversible.

polylysine

Polyalanine

that the structure

that the mercuric

some type ofcross-link

by Schulman

the viscosity with further

increases

increase

behaves

of polyalanine

in a

ion interacts

and Rideal normally

of surface

(ref. 97).

an unusual

effect

As the mixed

film

but then goes through presssure,

to the proteins

and lecithin.

acid

(cf. ref. 96).

into protein monolayers,

similarly

of the monolayer

was unaffected

acid were affected

groups on the protein or polyamino

are incorporated

first reported

for mixed monolayers

This suggested

and amino

forming

studied).

and polyglutamic

being

as shown in Fig. 11

The unusual

must be changing

a maximum

the behavior

behavior

shows

as it is compressed.

It

Fig. 11. Log ns vs. II for mixed monolayers of poly-DL-alanine and lecithin at pH 5.5; (0): polyalaning; (0): polyalanine-lecithin 4:l by weight; (A)polyalanine-lecithin 2:l by weight (from ref. 53).

378 was postulated between

that lipid molecules

the polypeptide

chains,

cause breaking

either

the net surface charge or by formation At low pressures comparatively however,

the effect

influence

VIII.

(high areas

becomes

the effect

bonds

increase of

bonds with the chains. is small because of the As the film is compressed,

of lipid molecules.

greater owing to the relatively

increasing

field of

of lipid molecules.

APPLICATIONS

In many natural

systems

(e.g., cell membranes,

the packing and conformation Monolayers

rect study. area occupied more,

hydrogen

steric effect,

of new hydrogen

per molecule),

low surface density

of keto-imido

by a simple

serve as excellent

per molecule

it can be varied

face pressure,

molecular

in a controlled

membranes

Membranes

have been studied. compartment

of protein monolayer

the

Further-

changes

may be followed.

in surA few ex-

studies

to practi-

here.

has been used by Verger and co-workers as surface

on the packing of membrane enzymes.

because

measured.

and the resulting

to di-

membranes

A direct approach spreadino

for these systems

and reactivity

applications

will be discussed

A. Biological

models

manner

conformation

dispersions),

is not easily amenable

at the surface can be precisely

amples of the many possible cal problems

protein-stabilized

of protein molecules

components

of intestinal

and catalytic

activity

trough by the Trurnit method

(ref. 5).

variables

of membrane-bound

brush border, human erythrocytes

They were spread from suspensions

cles, the amount of membrane

(ref. 98,99) by

films to study the effects of different

and -E. coli

of vesicles

on a multi-

Using 1251-labelled

vesi-

present at the surface was evaluated.

For intes-

tinal brush border membranes,

this was 25- 30% of the total amount

spread when

spreading

pressure

spread,

was at zero surface

the films were very stable.

proteins

spread from aqueous

and two brush border marker were assayed

was injected

and assayed.

tion of the experiment.

The specific

hydrolytic

the enzyme.

to that of pure

isotherms were measured and alkaline

phosphatase)

film by suction. out.

Films were com-

to a new compartment,

subphase,

Aliquots

No detectable

(i.e., no desorption)

the intact vesicles,indicating

is similar

were also carried

into the stirred

into the subphase

did not damage

activity

rinsed and transferred

were taken at intervals activity

Compression

(aminopeptidase

after removal of the surface

to 15 mNm_l,

substrate

This behavior

solution. enzymes

Kinetics of aminopeptidase pressed

and only 11% at 21 mNm -' (ref. 98). Once

activity

that spreading

of the subphase

release of aminopeptidase

was observed

during

the dura-

was in the same range as that of and subsequent

film manipulations

These results agree with the hypothesis

unit of the aminopeptidase

where the

is on the outer

that the

layer of the brush border

379 membrane border

When papain or trypsin was injected

(ref. 100).

film, a rapid release

occurred

with no change

Higher spreading cytoplasmic

portions

in surface

yields

of the two radioactive indicated

labels were recovered

the A/W interface

by the authors

In addition

functions

to this direct

coefficients

a dispersion

and cholesterol,

are important

of membranes

mixed

for membranes,

for synthetic

film

support

of cells at

the influence

of mem-

of the membrane.

the magnitude

on a solution

in the resistance

and

Fleming -.--* et al of interfacial

These were formed by spreading

membranes,

Diffusion

films of proteins

For example,

theory to calculate

of phosphatidylcholine

tion of calcium barriers

diffusion

This im-

in this work strongly

for studying

type of approach,

have been studied as models applied

reported

from the film.

there is no preferential

that the spreading

could prove a simple method

brane packing on biological

transfer

the hypothesis.

(33- 37%) and -E. coli

ratio in the surface

and therefore

Results

of either component.

the view expressed

(ref. 101)

with erythrocytes

that the protein/lipid

is the same as in the native membrane

lipids

brush phase

phospholipids

portant observation

spreadinq

under a spread

into the aqueous

further confirming

pressure,

were obtained

activity

When E. Coli cytoplasmic membranes containing (55- 60%). 14 -C-labelled proteins were spread, similar proand

membranes

32P-labelled

of aminopeptidase

of BSA followed

analysis

to passive

indicated transport

by the addi-

that interfacial across

these mem-

branes.

B. Foams and emulsions A dispersion

of a gas or liquid

stance

is present which adsorbs

persed

phase against

their capacity

Proteins

(ref. 102,103).

is unstable

unless a sub-

and thereby

stabilizes

compounds

vary greatly

Surface active

izers of foams and O/W emulsions. reviews

liquid

at the interface

coalescence.

as stabilizers.

in another

are particularly

effective

the disin

as stabil-

This has been the subject of some recent

The present

of how studies of protein monolayers

discussion

will be limited

may give information

to considerations

relevant

to these dis-

persed systems, When two emulsion

droplets

This means that during stabilizing

the coalescence

there is a reduction process

film and this could conceivably

at least in the initial film to compression

stage

would

(of the form rAdn).The

devised

(ref. 106) to measure and this parameter

ties of different

the work opposing

has been correlated

surfactants,

including

The response

the magnitude

tensiolaminometer

in interfacial

there is a compression

create a barrier

(ref. 104,105).

then determine

barrier

lamellae

coalesce,

of the interfacial

of the activation

reduction

energy

that has been of liquid

with foam stabilizing

proteins.

of the

to coalescence.

is an instrument surface

area.

proper-

A low compressibility

and

380 a low tendency teins,

to desorb

short-circuiting

to a great extent pressibility

both favor a high energy of the compression

(Part VI) although

of the monolayer.

case for colliding

droplets),

This is because

the relatively

displacement

measurements

relaxation

long times involved

The pushing out of segments

droplets

from the free energy

protein

segments

108).

protein,

tial at the surfaces effective

of droplets. agents

net charge,

(see

completion

barrier

during

of loops and

between

segments

produce a steric barrier a rise in concentration

flocculation

from the isoelectric

however,

(to coalescence)

proteins

(ref.

point of poten-

seem to be most

under conditions

that factors other

reof

of loops and tails can

thus promoting

is set up by the electrical

Generally,

showing

(formation

The presence

by molecules,

a repulsive

as stabilizing

carry a minimum

towards

The interaction

If the pH of the aqueous phase is displaced

the stabilizinq

be the

is quite

a high compressibility

phase

will usually

(ref. 107).

of droplets

the com-

(as would

in these measurements.

increase accompanying

in this region

also lead to bridging

increases

however

proceed

into the aqueous

as they approach

sulting

does not occur

of protein monolayers

indicate

processes

tails) by the protein could have two effects. on adjacent

of segments

the compressibility

case of pro-

In the

by desorption

For rapid compressions,

low, even though film balance Fiq. 3).

barrier.

barrier

where

than electrical

they repulsion

are predominant. The tendency

for proteins

to form thick films by surface coagulation

needs to be taken into account. surface

coagulation

This is particularly

for coalescence

be promoted

because

coagulation

in emulsion

stability

the well known phenomenon

of these films.

has not been studied

of phase inversion

a W/O one by shaking as in butter-making)

droplets

(Part IV-D).

may

The role of surface systematically

(conversion

is usually

as

from approach-

On the other hand, flocculation

to occur.

of the "stickiness"

preventing

also

to emulsions

occurs more readily at O/W than A/W interfaces

Thick films can form a simple steric barrier, ing close enough

relevant

although

of an O/W emulsion

attributed

to

to surface coag-

ulation of the protein at the A/W interface.

C.

Biomedical

problems

It is generally

accepted

ing recognition

and adhesion

role of surface

chemistry

part played by proteins and Freeman

(ref. 109).

drugs and hormones high molecular

weight

reactions

surface

functions)

in the biology

attached

is mediated

at the cell surface

Phagocytosis

glycoprotein,

of the living cell

by the cell surface.

of pinocytosis

has been discussed

is relevant.

the

for

such as the

(ref. 112) and surface

(ref. 113) are some of the many areas where

behavior

The

by Brandt

receptors

and the role of agents

fibronectin

(includ-

and, in particular,

(ref. 110), cell surface

(ref. ill), cell adhesion

blood-clotting of protein

that much of the behavior

induced

knowledge

381 An example of a natural appears

surfactant-covered

preventing

reduces

their collapse

been challenged liquid

in which monolayer

by Hills

surface

to cover the alveolar

tension,

at decreased

interface.

The major component

dipalmitoyl

lecithin,

adsorption

but other components

proteins

for the alveolar

preted the reproducible processes

dimensional in yield

hysteresis

at a molecular

level.

including

behavior

viscosity

The

as well as

studies

have

of the lung surfactant (ref. 117) have inter-

in terms of collapse

Blank and Britten

especially

proteins.

proteins

chemical

hysteresis

flow of films of lung surfactant

stress and surface

is phospholipid,

Mehta and Nagarajan

system.

for a discon-

at the solid tissue/air

serum-type

Surface

(ref. 115,116).

tended to focus on the expansion-compression as a model

the air spaces by

occurring

are present,

and comprises

A

of the lung.

The model has, however,

of the lung surfactant

is heterogeneous

surfactant-specific

cavities

thus stabilizing

lung volumes.

at an interface apparatus.

(ref. 114) who has put forward arguments

lining with surfactant

protein component

behavior

role is that of the respiratory

film is believed

This considerably

tinuous

system

to play an important

and reentry

(ref. 118) have measured

two-

and shown that there is a decrease. The results were interpreted

with age.

to indicate

that a two-dimensional network is formed rapidly having a yield -1 stress of about 3 mNm . The different processes of loop formation, desorption

and surface

coaqulation

are now better tribution

IX.

understood

of proteins

CONCLUDING Progress

during compression and should

for spreading

our knowledge.

valid method

At higher

urational

changes,

processes

relate

in conformation

desorption

to problems

under compression

may serve as a model

behavior

of the con-

films has been improved shows promise

in the very low pressure weights

providing

the different

posed in natural of molecules

(ultimately

membrane

parts of these molecules ing a configuration

of surface

appropriate

processes

leading

precautions

of molecular

For example,

to desorption

solution

that certain

These

the changes from the

the removal

conformation.

of The

has.not yet

segments

could form loops in the non-aqueous

that would make the probability

config-

of whole molecules)

biosynthesis:

under high compression

It seems possible

for fur-

provide a

have ben delineated.

systems.

to take up its unique proteins

region

(tails and loops) are expelled

for the final step in protein

in detail.

facets of protein monolayers.

and surface coagulation

from the ribosome

of integral

been studied

understanding films.

such as spectroscopy

molecular

pressures,

as portions

certain

and manipulation

Measurements

for determining

are observed.

(see Fig. 7)

REMARKS

of newer techniques

the molecule

in lipid-protein

has been made in understanding

The methodology

surface

lead to a better

to hysteresis

and application thering

of protein monolayers

in the apolar

phase, thus creat-

of desorption

extremely

low.

382 Direct studies of the desorption esting

results.

surface

of other proteins

One is that smaller

proteins

by those of higher molecular

ations where

there is competition

such as in blood clotting. protein

Another

reached.

between

is that desorption

different

to situ-

species,

of a slightly

soluble

at the same rate as from the pure monolayer

until low surface coverages

This is pertinent

from the

a result that is relevant

for the surface

from a mixed film proceeds

at a given pressure

weight,

have led us to some inter-

tend to be displaced

to considerations

of the desorbing

of mass transport

protein are across

biological

membranes. The structure

of protein monolayers

the newer spectroscopic

techniques

should help to clarify

the details.

compressibilities plained.

of different

functions

is warranted.

are studied;

photosynthetic

proteins which

in Photosystem

Processes

such as surface

size but dependent

to reflect

flow and loop formation

the surface

for proteins,

from the surface

projects

mainly apolar

secondary

structure

Because

strongly

ular to its axis. of proteins,

in the protein)

can act as

side-chains

to be

favor a random

from protein and polypeptide

the presence

of the a-helix

there is a tendency

in

for one surface

The mean helical

of polar and apolar

the retention hydrophobic

surfaces,

that the helix is highly amphiphilic

Krebs and Phillips

(ref. 121,122)

between

the product

in the molecule

of

moment,

a large perpendic-

have found, for a number ;hF (u,, being the average

and F being the fraction at the A/W surface

of a-helix

for a given amount

into the subphase.

The design of multicompartment of reactions

appear

concept

and the value of II exerted

injected

of

on the

This has led to the interesting

of the separation

value of p,, for all helixes

of protein

an A/W interface

(ref. 120j which justifies

monolayers.

a good correlation

consisting,

surface

moment

meaning

to be independent

phase, conditions

support

accessory

polar side chains while the opposite

side chains.

in protein

value for the parameter

ex-

(ref. 119).

flow and loop formation

polar and apolar

In some proteins,

hydrophobic

Ph, gives a measure

reactions

Surface

allowing

of each helix to project mainly

of the helical

appear

On the other hand, evidence

conformation.

an essentiaT and red algae

on a segment of the molecule

towards and away from the aqueous

films removed

in situ -in the

to amino acid composition

is phycocyanin,

a random chain conformation.

chain configuration.

variations

have never been satisfactorily

are related

2 of blue-green

of 6- 10 amino acid residues.

a "good" solvent

although

of monolayers

There are some marked

a recent example

pigment

examination

resolved

This becomes more so as new proteins with specific

molecular

directed

for direct

A study of how these variations

and structure

average,

is still not entirely

in protein

troughs

monolayers.

to date but other

processes

has provided

a stimulus

They have been used mainly are amenable

to study,

to the study

to follow enzyme

including

many in

383 the biological approach. in membrane

X.

area, which would benefit

This includes mimetic

from a collaborative

the further development

of protein

multidisciplinary monolayers

as a tool

chemistry.

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