NON-IONIC DETERGENTS

Non-ionic Detergents

2

NON-IONIC DETERGENTS OFFERED BY BACHEM

Detergents are amphipathic molecules that contain both

polar and hydrophobic groups. All detergents are char-

acterized as containing a hydrophilic “head” region and

a hydrophobic “tail” region. In contrast to purely polar

or non-polar molecules, amphipathic molecules exhibit

unique properties in water. Their polar group forms hydro-

gen bonds with water molecules, while the hydrocarbon

chains aggregate due to hydrophobic interactions. These

properties allow detergents to be soluble in water and also

to solubilize hydrophobic compounds in water.

NON-IONICDETERGENTS

Bachem offers alkanoyl-N-methyl-

glucamides (MEGA’s), alkylglycosides,

and oligoethyleneglycol monoalkyl

ethers. Besides these types of non-

ionic detergents, a choice of lipids and

phospholipids can be ordered at our

online shop: shop.bachem.com

3

Overview Detergents

Detergents are used in biochemistry to

solubilize membrane-bound proteins and

facilitate their isolation and recrystalliza-

tion [1,2]. They can be used to introduce the

purifi ed protein molecules into lipo-

somes and to determine their properties

or enzymatic activities in reconstitution

experiments [3,4]. Detergents can further

be used to facilitate protein solubilization in

biological assays or to form microemulsions

for electrochemical studies [5].

There are hundreds of detergents that can

be used in biochemical experiments. The

selection of the “right” detergent for a given

application is therefore diffi cult and still

rather empirical [1,3,6,7].

The following physical properties of deter-

gents can help making the right decision.

Critical Micelle Concentration (CMC)

The CMC of a detergent is the minimum

concentration at which micelles form. The

CMC is also an indicator of the strength

detergents bind to proteins; i.e., low values

indicate strong binding and high values

indicate weak binding.

The CMC is infl uenced by pH, temperature,

ionic strength and impurities in the solution

[1,7,8]. The CMC values reported in litera-

ture (e.g. [9]) are therefore only correct for

the given conditions.

• A high CMC is desirable when dialysis

across a membrane is necessary and in

other situations where rapid removal or

displacement of detergent is desired.

• A low CMC is desirable if the ratio of free

to bound detergent has to be minimized, e.g.

in the measurement of binding strength of

detergent to protein.

• The CMC of a given detergent can be

determined by different methods including

the measurement of light scattering, sur-

face tension, hydrodynamic properties, and

changes in absorbance or fl uorescence

upon dye solubilization [10].

Aggregation Number (N)

The aggregation number is the average

number of detergent monomers in one

micelle and permits the determination of

the micelle size and the molecular weight.

The micelle size is important in gel fi ltration

experiments since the separation of differ-

ent proteins according to size is done more

easily in the presence of a detergent with a

small micellar size [6]. This value is depen-

dent on temperature and ionic strength.

Cloud Point (cp)

The cloud point is the temperature above

which detergent micelles form super-

aggregates and the solution separates in a

solvent-rich phase and a solvent-depleted

phase [7]. This phase separation can be

exploited for protein extraction.

CnE

mProduct

C5E

1Ethyleneglycolmonopentylether

C5E

2Diethyleneglycolmonopentylether

C5E

3n-Pentyltrioxyethylene

C6E

1Ethyleneglycolmonohexylether

C6E

3n-Hexyltrioxyethylene

C6E

4n-Hexyltetraoxyethylene

C6E

5n-Hexylpentaoxyethylene

C7E

3n-Heptyltrioxyethylene

C7E

4n-Heptyltetraoxyethylene

C7E

5n-Heptylpentaoxyethylene

C8E

1Ethyleneglycolmonooctylether

C8E

3n-Octyltrioxyethylene

C8E

4n-Octyltetraoxyethylene

C8E

5n-Octylpentaoxyethylene

C8E

nn-Octylpolyoxyethylene

C10

E4

n-Decyltetraoxyethylene

C10

E5

n-Decylpentaoxyethylene

C12

E5

Dodecyl pentaethylene

glycolether

n-Alkyloligooxy-

ethylenes

(Oligoethyleneglycol

monoalkyl ethers)

CnE

m: C

n = CH

3(CH

2)

n-1

alkyl chain

Em

= (OCH2CH

2)

mOH

oligoethyleneglycol

In particular, the octyl

oligooxyethylenes C8E

4,

C8E

5, and C

8E

n (Octyl-

POE, n = 2 to 9) have

been shown to be of

great value for solubili-

zation and crystalliza-

tion of proteins, such as

porin, from E. coli outer

membranes.

Non-ionic Detergents

4

Critical Micellar Temperature (cmt)

The critical micellar temperature is defi ned

as the minimum temperature at which a

detergent can form micelles in water. At

temperatures below cmt, some detergents

exist as insoluble liquid crystals. This pa-

rameter has to be taken into consideration

in protein purifi cation at low temperatures.

Classifi cation

Detergents can be grouped in four main

classes according to the properties of their

head group:

• Anionic

• Cationic

• Zwitterionic (Ampholytic)

• Non-Ionic

Anionic and cationic detergents typically

modify protein structure to a greater extent

than the other two classes.

Zwitterionic detergents are unique as they

offer the combined properties of ionic and

non-ionic detergents. Like non-ionic deter-

gents the zwittergents do not possess a

net charge, they lack conductivity and

electrophoretic mobility and do not bind

to ion-exchange resins. However, like ionic

detergents, they are effi cient at breaking

protein-protein interactions.

The group of non-ionic detergents, pre-

sented in this brochure, is special in the

sense that they can be classifi ed as “mild”

detergents because they are less

likely to denature proteins than ionic deter-

gents. On the other side they are less effec-

tive at disrupting protein aggregation.

The most important properties of non-ionic

detergents are:

• Uncharged hydrophilic head group.

• Better suited for breaking lipid-lipid and

lipid-protein interactions.

• Considered to be nondenaturants.

• Salts have minimal effect on micellar size.

• Solubilize membrane proteins in a gentle

manner, allowing the solubilized proteins to

retain native subunit structure, enzymatic

activity and/or nonenzymatic function.

• The CMC of a non-ionic detergent is

relatively unaffected by increasing ionic

strength, and increases substantially with

rising temperature.

The amphipathic

nature of detergents is

shown as an example

with n-Dodecyl-β-

D-maltoside (DDM,

Product P-1170).

Maltose constitutes

the hydrophilic head

and the alkyl chain the

hydrophobic tail.

Hydrophilic head

Hydrophobic tail

MILD NON-IONIC DETERGENTS FOR THE SOLUBILIZATION OF MEMBRANE-BOUND PROTEINS

5

Practical Considerations

Denaturation Effect

When characterizing a protein in its native

state and studying its functions, the dena-

turation effect of detergents is an important

point. It is diffi cult to classify detergents into

denaturizing and non-denaturizing classes

and to ascribe these properties to specifi c

features of either the monomeric or the

micellar structure of the molecule [8]. The

denaturation effect of a detergent depends

further on the structure of the protein itself.

The following general statements can be of

practical use:

• Non-ionic detergents with polyoxyethylene

or sugar head groups do usually not dena-

turize proteins.

• Ionic detergents are nearly always denatur-

ants at temperatures and concentrations

used for complete membrane solubilization.

They further dissociate complex proteins in

their polypeptide chains. This effect may be

useful in the separation and identifi cation of

the different subunits of a protein [6].

Detergent Amount / Concentration

The appropriate amount of detergent (con-

centration) must be used for a successful iso-

lation of a protein. The membranes undergo

different stages of disintegration with an

increasing amount of detergent:

• At concentrations of around 0.1 mg to 1 mg

detergent per mg membrane lipid, selective

extraction of membrane proteins can occur

but the membrane bilayer remains essentially

intact.

• At higher concentrations of about 2 mg

detergent per mg lipid, solubilization of

the membrane occurs. This results in the

formation of soluble lipid-protein-detergent,

protein-detergent and lipid-detergent mi-

celles [1].

• 10 mg detergent per mg lipid or more should

be used for delipidation (i.e. a maximal ex-

change of the lipid bound to the protein with

detergent). At this point protein-detergent mi-

celles are formed, each containing essentially

only one protein molecule. These micelles can

then be separated by methods based on size,

charge density, binding affi nity and solvent

partitioning [11].

In the solubilization process the binding of

detergent to protein or membranes (solubi-

lization) has to compete with the self-asso-

ciation of detergent molecules to micelles

[1,12]. The exact amount of detergent

needed to achieve a certain effect depends

on the CMC, the micelle size, the tempera-

ture, the nature of the membrane and the

detergent [1]. The detergent monomers do

not participate in membrane solubilization,

but they are required to obtain the mono-

mer-micelle equilibrium [11].

To calculate the effective detergent-to-lipid

ratio and the amount of detergent available

for solubilization, the amount of detergent

forming micelles must be taken into

account:

• Detergents with low CMC; the effective

amount of detergent essentially equals the

total amount of detergent added, since very

little detergent exists as monomers.

• Detergents with a high CMC; the effec-

tive amount of detergent equals the total

amount of detergent added minus the

monomer concentration (essentially the

CMC).

References

[1] A. Helenius and K. Simons, Biochim.

Biophys. Acta 415, 29 (1975)

[2] R.M. Garavito and J.A. Jenkins, Structure

and Function of Membrane Proteins (E.

Quagliariello and F. Palmieri, eds.), Elsevier

Science Publishers B.V., Amsterdam (1983)

[3] C. Tanford and J.A. Reynolds, Biochim.

Biophys. Acta 457, 133 (1976)

[4] M. Hanatani et al., J. Biochem. 95, 1349

(1984)

[5] M.O. Iwunze et al., Anal. Chem. 62, 644

(1990)

[6] A. Helenius et al., Methods Enzymol. 56,

734 (1979)

[7] J.M. Neugebauer, Methods Enzymol. 182,

239 (1990)

[8] L.M. Hjelmeland, Methods Enzymol. 124,

135 (1986)

[9] P. Mukerjee and K.J. Mysels, National

Standards Reference Data Series, Vol. 36,

US National Bureau of Standards (NSRDS-

NBS 36), Washington (1971)

[10] A. Chattopadhyay and E. London, Anal.

Biochem. 139, 408 (1984)

[11] D. Lichtenberg et al., Biochim. Biophys.

Acta 737, 285 (1983)

[12] C. Tanford, J. Mol. Biol. 67, 59 (1972)

Despite the large num-

ber of detergents that

are commercially avail-

able, no single “univer-

sal detergent” is ideally

suited to all biochemi-

cal applications.

(G.G.Privé)

Non-ionic Detergents

6

C. Tanford

The hydrophobic effect: Formation

of micelles and biological mem-

branes.

Second edition.

John Wiley and Sons, New York

(1980)

U. Pfüller

Mizellen - Vesikel- Microemul-

sionen.

Tensidassoziate und ihre Anwend-

ung in Analytik und Biochemie.

Springer-Verlag, Berlin (1986)

H. Michel

Crystallization of membrane pro-

teins.

CRC Press Inc., Boca Raton (1991)

E.D. Goddard and K.P. Ananthapad-

man-anbhan, eds.

Interactions of surfactants with

polymers and proteins.

CRC Press Inc., Boca Raton (1993)

K. Holmberg, ed.

Novel surfactants: Preparation, ap-

plications and biodegradability.

Second edition.

Marcel Dekker Inc., New York (2003)

GENERAL REFERENCESM. Caffrey

Membrane protein crystallization.

J. Struct. Biol. 142, 108-132 (2003)

M.C. Wiener

A pedestrian guide to membrane

protein crystallization.

Methods 34, 364-372 (2004)

D. Myers

Surfactants science and technology.

Third edition.

John Wiley and Sons, New York

(2006)

G.G. Privé

Detergents for the stabilization

and crystallization of membrane

proteins.

Methods 41, 388-397 (2007)

7

NON-IONIC DETERGENTS

Bilayers and Micelles.

Detergents are amphipathic molecules

that contain both polar and hydrophobic

groups. All detergents are character-

ized as containing a hydrophilic “head”

region and a hydrophobic “tail” region.

In contrast to purely polar or non-polar

molecules, amphipathic molecules

exhibit unique properties in water. Their

polar group forms hydrogen bonds with

water molecules, while the hydrocarbon

chains aggregate due to hydrophobic

interactions. These properties allow de-

tergents to be soluble in water and also

to solubilize hydrophobic compounds in

aqueous systems.

Non-ionic Detergents

8

ALKYL GLYCOSIDES

MEGA-8

(Octanoyl-N-methylglucamide)

P-1060

MEGA-9

(Nonanoyl-N-methylglucamide)

P-1165

MEGA-10

(Decanoyl-N-methylglucamide)

P-1000

MEGA-12

(Dodecanoyl-N-methylglucamide)

P-1175

n-Nonyl β-D-glucopyranoside

(NGP)

P-1150

Octyl glucoside

(OGP)

P-1110

n-Dodecyl-β-D-maltoside

(DDM)

P-1170

N-D-GLUCO-N-METHYL-ALKANAMIDES(MEGAs)

NON-IONIC DETERGENTSAlkanoyl-N-methylglucamides combine a high solubilization power with non-denatur-

izing properties. They don’t interfere with the photometric monitoring of proteins at 280

nm (as their absorption maximum lies at 220 nm). Their easy removal by dialysis makes

them valuable tools for membrane studies. Like the alkanoyl-N-methylglucamides, the

alkylglucosides are mild detergents. Their CMC are only slightly affected by variations

of ionic strength. Oligoethyleneglycol monoalky ethers are standard detergents for the

solubilization and structural characterization of integral membrane proteins.

9

OLIGOETHYLENE-GLYCOL MONOALKYL ETHERS AND SULFOXIDES

Ethyleneglycolmonopentylether

(C5E

1; n-Pentylmonooxyethylene)

P-1055

Diethyleneglycolmonopentylether

(C5E

2; n-Pentyldioxyethylene)

P-1025

n-Pentyltrioxyethylene

(C5E

3; Triethyleneglycolmonopentyl

ether)

P-1135

n-Hexyltrioxyethylene

(C6E

3; Triethyleneglycolmonohexyl ether)

P-1095

n-Hexyltetraoxyethylene

(C6E

4; Tetraethyleneglycolmonohexyl

ether)

P-1085

n-Hexylpentaoxyethylene

(C6E

5; Pentaethyleneglycolmonohexyl

ether)

P-1080

n-Heptyltrioxyethylene

(C7E

3; Triethyleneglycolmonoheptyl

ether)

P-1075

n-Heptyltetraoxyethylene

(C7E

4; Tetraethyleneglycolmonoheptyl

ether)

P-1070

n-Heptylpentaoxyethylene

(C7E

5; Pentaethyleneglycolmonoheptyl

ether)

P-1065

Ethyleneglycolmonooctylether

(C8E

1; n-Octylmonooxyethylene)

P-1050

n-Octyltrioxyethylene

(C8E

3; Triethyleneglycolmonooctyl ether)

P-1125

n-Octyltetraoxyethylene

(C8E

4; Tetraethyleneglycolmonooctyl

ether)

P-1120

n-Octylpentaoxyethylene

(C8E

5; Pentaethyleneglycolmonooctyl

ether)

P-1115

n-Octylpolyoxyethylene

(C8E

n (n = 2 to 9); Octyl-POE;

Rosenbusch-Tenside)

P-1140

n-Decyltetraoxyethylene

(C10

E4; Tetraethyleneglycolmonodecyl

ether)

P-1010

n-Decylpentaoxyethylene

(C10

E5; Pentaethyleneglycolmonodecyl

ether)

P-1005

Dodecyl pentaethyleneglycolether

(C12

E5; Pentaethyleneglycolmonododecyl

ether)

P-1160

rac-2,3-Dihydroxypropyloctylsulfoxide

(n-Octyl-rac-2,3-dioxypropyl sulfoxide)

P-1040

2-Hydroxyethyloctylsulfoxide

(n-Octyl-2-hydroxyethyl sulfoxide)

P-1105

Non-ionic Detergents

10

Product

Number

Product CMC

(mM)

Conditions N (H2O) References

P-1060 MEGA-8 73 25°C F. Yu and R.E. McCarty, Arch. Biochem. Biophys. 238, 61 (1985)

P-1165 MEGA-9 25

50

25°C

15°C

V. de Pinto et al. , Eur. J. Biochem. 183, 179 (1989)

P-1000 MEGA-10 7 M. Hanatani et al., J. Biochem. 95, 1349 (1984)

P-1175 MEGA-12 0.35 25°C Y.-P. Zhu et al., J. Surf. Det. 2, 357 (1999)

P-1150 n-Nonyl β-D-glucopyranoside

6.5

3.5

7.5

low ionic strength

1M NaCl

25°C

133 W.J. de Grip and P.H.M. Bovee-Geurts, Chem. Phys Lipids 23, 321 (1979)

F. Yu and R.E. McCarty, Arch. Biochem. Biophys. 238, 61 (1985)

P-1110 Octyl glucoside 23.2

13.5

25.0

low ionic strength

1M NaCl

25°C

8427-100

W.J. de Grip and P.H.M. Bovee-Geurts, Chem. Phys Lipids 23, 321 (1979)

K. Shinoda et al., Bull. Chem. Soc. Jpn. 34, 237 (1961)

P-1170 n-Dodecyl-β-D-maltoside

170 78-14998

W.J. de Grip, Methods Enzymol. 81, 256 (1982)J. Kern et al., Photosynth. Res. 84, 153 (2005)

P-1095 n-Hexyltrioxyeth-ylene

100 25°C P. Becher, Micelle formation in aqueous and nonaqueous solutions. In: Nonionic Surfactants, M.J. Schick, ed., Marcel Dekker Inc., New York, p. 478 (1967)P-1085 n-Hexyltetraoxyeth-

ylene90 20°C

P-1080 n-Hexylpentaoxyeth-ylene

90 20°C

P-1050 Ethyleneglycolmon-ooctyl ether

4.9 25°C

P-1125 n-Octyltrioxyeth-ylene

7.5 25°C

P-1120 n-Octyltetraoxyeth-ylene

12.4

3.6

6°C

60°C

82 M. Corti et al., Phys. Rev. Lett. 48, 1617 (1982)R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986)

P-1115 n-Octylpentaoxyeth-ylene

4.3 25°C M. Zulauf and J.P. Rosenbusch, J. Phys. Chem. 87, 856 (1983)

P-1140 n-Octylpolyoxyeth-ylene

6.6 R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986)

P-1010 n-Decyltetraoxyeth-ylene

0.98

0.68

10°C

25°C

53 ± 10 P. Becher, Micelle formation in aqueous and nonaqueous solutions. In: Nonionic Surfactants, M.J. Schick, ed., Marcel Dekker Inc., New York, p. 478 (1967)

P-1005 n-Decylpentaoxyeth-ylene

1.18

0.81

10°C

25°C

73

P-1105 2-Hydroxyethyloctyl sulfoxide

29.9 R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986)

CALCITONINGENE-RELATEDPEPTIDES

ANTIMICROBIALPEPTIDES

AMYLOID PEPTIDES

CASPASESUBSTRATES INHIBITORS

CYSTEINEDERIVATIVES

GHRELIN,LEPTIN ANDOBESTATIN

1

DAP AND DABDERIVATIVES

ENDOTHELINS

1

FRET SUBSTRATES

DIABETES PEPTIDES

PEPTIDES IN COSMETICS

PARACTIVATINGPEPTIDES

NON-IONIC DETERGENTS

ORTHOGONALITYOF PROTECTINGGROUPS

PEPTIDE YY

VETERINARYPEPTIDES

PRIONPEPTIDES

PSEUDOPROLINEDIPEPTIDES

SECRETASESUBSTRATES INHIBITORS

VIP/PACAP

MATRIXMETALLO-PROTEINASES

LHRHAGONISTS AND ANTAGONISTS

MELANOMA PEPTIDES

NEUROPEPTIDE YN-METHYLATEDAMINO ACIDDERIVATIVES

PRODUCT BROCHURES

2006

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