Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels

Vol. 28, No. 5, 1967

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MOLECULARWEIGHT ESTIMATION OF POLYPEPTIDE CHAI S BY ELECTROPHORESISIN SDS-POLYACRYLAMIDEGELS!f Arnold

Department of Ophthalmology and Department of Biochemistry Eladio Vi%ela2, New York University School of Medicine New York, New York and Jacob V. Maizel, Jr.3, Department of Cell Biology Albert Einstein College of Medicine Bronx, New York

Received

August

L. Shapiro,

3, 1967

Introduction. Electrophoresis detergent

sodium dodecyl

separation et al., --

in polyacrylamide

and identification 1966;

to indicate estimation

Vi'iiuela that

the

in the presence

(SDS) has proven of polypeptide

et al., --

It

1967).

same technique

of the molecular

Materials

to be a useful

chains

(Maizel,

is the purpose

may be used for

weights

of proteins

of the anionic

1966;

of this

the rapid

and their

tool

for

A, lysozyme,

from the Worthington was a gift and trypsin and bovine

Biochemical

from Dr. R. Warner; from Calbiochem. serum albumin

(H and L dimer)

Shapiro

communication and simple

subunits.

carboxypeptidase Corporation. bovine

from Armour.

Ovalbumin

hemoglobin

Thyroglobulin

were produced

A, and pepsin

Half

type

were obtained

(3x crystallized)

was purchased II

molecules

from Pentex

was obtained

from Sigma

of gamma globulin

by the MPC-11 mouse plasma cell

tumor

(Scharff

1967).

1Supported by USPHS Grants NB-U6743, AI-4153, and AM-01845, International Post-Doctoral Fellowship F05-TW-84g2, NSF Grant GB-4751, and grants from the American Cancer Society. 2Present address: Instituto Mara%on, Centro De Investigaciones Biol;gicas, Veld'zquez 138, Madrid 6, Spain. 3Recipient

the

and Methods.

Ribonuclease

--et al.,

sulfate

gels

of USPHS Career

Development

815

Award.

Vol. 28, No. 5, 1967

Fifty

BIOCHEMICAL

microgram

AND BIOPHYSICAL

samples of each protein

pH 7.1 in 1% SDS and 1% 2-mercaptoethanol samples were then

dialyzed

16 hours

pH 7.1 with

(H) and light

result

study.

(L)

Iodoacetamide of disruption

The gels

molecular tion

chains

(0.05M)

pH 7.1 with for

0.l.M phosphate

and

was O.lM phosphate

gamma globulin

against

and tertiary

O.OlM phosphate

in 20% sulfosalicylic 5 hours,

0.1% SDS but without acid

for

and destained

with

correlated

16 hours,

with

studies

performed

in electrophoretic under

these

of relative

conditions

This 2-Me.

acid.

within

The mig-raeach gel. and beta

migration.

on some of these

migration

as a

the known

for each protein.

the migration

with

stained

7% acetic

of the alpha

as a standard

exposed

by the SDS.

tabulated,

In preliminary

that

sulfhydryls

point

using

heavy

but was incubated

structure with

1959) were

above to yield

blue was used as the reference

of hemoglobin

no differences

(Fahey,

as described

migration of each band was then weights 4 of the polypeptide chains

were then

suggesting

buffer

at The

at 8 volts/cm.

containing

was added to block

of secondary

were fixed

of bromphenol

The data

3 hours.

O.OlM phosphate

The second was not reduced

0.25% Coomasie Blue for

The relative

rabbit

One was treated

chains.

sample was dialyzed

with

and reduced

0.1% SDS.

in this

1% SDS.

gels

The electrophoresis

1966).

Two samples of DEAE-purified included

against

at 37' for

was performed

in 125 m.m. 5% polyacrylamide

0.1% SDS (Maizel,

were denatured (2-Me)

Electrophoresis

0.1% SDS and 0.1% 2-Me. two hours

for

RESEARCH COMMUNICATIONS

in O.l%,

of incubation

proteins,

we found

O.S%, and 1.0% SDS, and dialysis,

0.1%

SDS is sufficient.

4The molecular weights used were obtained from The Proteins, ed. H.Neurath (New York and London: Academic Press, 1963); references listed in the Worthington Biochemical Corp. catalog; Edelhoch, H., and De Crombrugghe, B., J. Biol. Chem., 241, 4357 (1966); and Fleischman, J. B., Ann. Rev. Biochem., -35, 835 (1966). -

816

BIOCHEMICAL

Vol. 28, No. 5, 1967

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Results. A typical in Fig.

experiment

performed

under

the

conditions

described

is

shown

1.

Ribonuclease I I I

A

n

Lysozyme Thyroglobulln

J +

Hemoglobin

n

m Trypsin

1

1

n H

+

L

1

Chains mm

1

1

Carboxypept

idase

A

I

I

I

Pepsin I

I I I

Bovine :;

1 Serum

Albumin

n

J

Gamma 1

I

Ovalbumin ii

Globulin

I

I

Figure 1 - Stained 5% polyacrylamide disc gels prepared and electrophoresed as described in the text to show the relative migrations of the polypeptide The anode is on the right. The bands of chains of a number of proteins. bromphenol blue are not shown in this illustration.

Both ovalbumin component.

Carboxymethylation

of the slow component by disulfide

and bovine

bonding

serum albumin of reduced

in each case, during

revealed

samples

suggesting

electrophoresis

81’7

that

a second,

resulted

in disappearance

aggregation

had occurred.

slow moving

into

dimers

Vol. 28, No. 5, 1967

BIOCHEMICAL

z

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Trypsin L Chain

20

101 ’

’

(Origin)

’

2o

’

’

’

4o

’

6o

RELATIVE

’

’

’

*O

‘OO(&,j

MIGRATION

Figure 2 - Semi-log plot of molecular weight against distance of migration A line of best fit has relative to the alpha and beta chains of hemoglobin. The insert represents a rectilinear plot been drawn through the data points. of the same data.

A composite migration

of three

is plotted

straight

line

relationship

such experiments

against

the

can be fitted between

is shown in Fig.

log of molecular

from molecular

molecular

weight

weight

weights

15,500

and migration

2.

If

relative

(ordinate),

a

to 165,000.

can thus

The

be expressed

as:

M.W. = k (lO-bx) where x is the distance The rectilinearly relationship of 15,000 weights

between to 75-90,000

studied,

A limited could

of migration plotted

rates for

however,

insert

in Fig.

of migration these the

true

way.

slope.

2 emphasizes

and molecular

5% gels.

indicated

almost

linear

in the range

range

of molecular

is hyperbolic. that

other

A 5% gel was chosen for

818

the

weights

Over the entire

function

number of experiments

be used in a similar

and b is the

gel concentrations illustration

because

Vol. 28, No. 5, 1967

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

many polypeptides

of biological

interest

within

linear

to 90,000

the almost The deviations

were repeatable,

15,000

of ribonuclease and,

as yet,

have molecular

weights

which

fall

range.

A and lysozyme

not completely

from the straight

line

explained.

Discussion. As suggested phoresis spite

by Smithies

can be a useful

the choice

that

proteins

as anions

disruption

and 2-Me results soluble

(e.g. Thus,

component

of a mixture

necessary and the during

ati

curves,

and that

formation disulfide

with

1, that

linkages

by SDS

of many relatively

the method

intechnique

parallel

the molecular simply

gel.

the intrachain

bonds disrupted

is as applicable as to a single

approximate

both

The

method of choice.

and hemoglobin)

in a second,

all SDS.

and the ease of the polyacrylamide

of unknown polypeptides

disulfide

differences

De-

points

the fitted

of complex

as the electrophoretic

to carboxymethylate interchain

charge

electro-

weight.

of isoelectric

approximated

solubilization

one can readily

of known size

of molecular

a range

hydrophobic,

thyroglobulin

or protein.

marker

the native

as the result

can be seen from Fig.

of proteins

with

points

in the quantitative

recommend it

It

all

These factors

and Humphrey (1959),

the approximation

of hydrogen,

proteins.

strongly

for

1953),

SDS minimizes

migrate

extensive

tool

and Allison

of a group of proteins

from 4 to 11 (Alberty, suggesting

(1962)

to a mixture polypeptide

weight

by running

In many cases, sulfhydryls

of each

a suitable it

may be

exposed

by 2-Me to prevent

by SDS

aggregation

electrophoresis. Finally,

the

can be increased

sensitivity, by combined

and a gel fractionator weights

of minute

gel (Shapiro,

(Maizel,

resolving

power,

use of radioactive 1966) to detect

amounts of newly

synthesized

in preparation).

819

and usefulness

of this

method

amino acids,

double

and estimate

the molecular

material,

all

within

labeling,

a single

Vol. 28, No. 5, 1967

Acknowledgements. We gratefully of Miss Janet

Reilly

acknowledge

the expert

technical

assistance

and Miss Jana Krausova.

References Alberty, R. A., (New York:

in The Proteins, Academic Press,

Allison,

and Humphrey,

A. C.,

Fahey, J. L., Maizel,

J. Biol.

J. V., Jr.,

ed. Neurath, H., and Bailey, 1953), vol. 1, part A, 511. J. H., Nature,

Chem., 234,

Science,

151,

2645,

Shapiro, Smithies,

A. L.,

183, 1590 (1959).

(1959).

988 (1966).

Scharff, M. D., Shapiro, A. L., Ginsberg, B., Symposium on Quantitative Biology, 1967, Shapiro, A. L., Proc. Nat.

Cold Spring in press.

Scharff, M. D., Maizel, J. V., Jr., Acad. Sci., -56, 216 (1966). in preparation.

O., Arch.

Vii?uela, E., Algranati, 2, 3 (1967).

Biochem. I.

K.

Biophys.,

Harbor

and Uhr, J. W.,

-98, Supp. 1, 125 (1962). D., Ochoa, S., Europ. J. Biochem.,

820