13
The Journal of Biochemijtry. Vol. 57. No. 3. 1965 The Interaction of Detergents with Proteins The Effect of Detergents on the Conformation of Bacillus subtilis a-Amylase and Bence-Jones Protein By AKIRA IMANISHI, YOSHIHIDE MOMOTANI* and TOSHIZO ISEMURA (From the Division of Physical Chemistry, Institute for Protein Research, Osaka University, Osaka) (Received for publication, November 25, 1964) Many investigations on the interaction of detergents with proteins and synthetic poly- mers have shown that detergents have a high affinity to macromolecules even at low deter- gent concentration (110). The detergents exert a wide variety of effects on proteins depending on the nature of protein under study and on such conditions as temperature, pH and ionic strength. A detergent molecule is amphipathic and consists of two distinctly distinguishable parts, namely, hydrocarbon chain of hydrophobic character and hydro- philic group. The hydrocarbon chasns associate with each other to form micelle above a con- centration called c.m.c.** It may be expected that the hydrocarbon chains penetrate into the hydrophobic fabric of a protein molecule to enhance conformational change. The con- formational change of the protein molecule has recently been investigated by the methods of optical rotatory dispersion (//—15) and ultraviolet absorption difference spectrum (16). J i r g e n s o n s found that an addition of detergents to proteins having a value of b a close to zero, e.g. ^-globulin, increased the value of b 0 and that the reduced and carboxy- methylated albumin showed a conformational dependent-Cotton effect in far ultraviolet region (1114). These findings suggest that detergent facilitates the formation of ordered structure which differs from that of the native molecule. While, it was found that, with increase in * Present address ; Tezukayama University, Nara. •• The abbreviation used; c.m.c, the critical micelle concentration. the concentration of anionic detergent added at pH 7, bovine plasma albumin showed an expansion which was very similar to isomeri- zation (17) caused by increasing acidity (15). The present investigation was undertaken to obtain further information on the mecha- nism of the action of detergent on protein conformation. The effects of three different kinds of detergents, iur., anionic, cationic and nonionic detergents on the conformation of two different proteins were studied as func- tions of pH and detergent concentration. One of the proteins is bacterial a-amylase [EC 3. 2. 1. 1, a-1, 4-glucan 4-glucanohydrolase. Bacil- lus subtilis), which has a folded structure including an a-helical structure without disulfide linkage. The other is Bence-Jones protein which is found in the urine of patient suffering from multiple myeloma. This protein has probably intrachain cross /S-structure but not a-helical structure as observed for y- globulin (18). The effect of detergent on the denatured a-amylase, in which a-helical struc- ture had been destroyed, was also examined. The experimental results showed that the original conformation of the protein was broken completely and that the unfolded polypeptidc chain assumed to take partially a-helical structure, content of which depended upon the pH. Both anionic and cationic detergents behaved as a helix-former even at extremely alkaline and acidic pH while nonionic detergent did not have any helix- forming ability in the pH range of interest. 417 Downloaded from https://academic.oup.com/jb/article-abstract/57/3/417/936916 by guest on 31 January 2018

The Interaction of Detergents with Proteins The Effect of Detergents

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The Journal of Biochemijtry. Vol. 57. No. 3. 1965

The Interaction of Detergents with Proteins

The Effect of Detergents on the Conformation of Bacillus subtilisa-Amylase and Bence-Jones Protein

By AKIRA IMANISHI, YOSHIHIDE MOMOTANI*

and TOSHIZO ISEMURA

(From the Division of Physical Chemistry, Institute for Protein Research,Osaka University, Osaka)

(Received for publication, November 25, 1964)

Many investigations on the interaction ofdetergents with proteins and synthetic poly-mers have shown that detergents have a highaffinity to macromolecules even at low deter-gent concentration (1—10). The detergentsexert a wide variety of effects on proteinsdepending on the nature of protein understudy and on such conditions as temperature,pH and ionic strength. A detergent moleculeis amphipathic and consists of two distinctlydistinguishable parts, namely, hydrocarbonchain of hydrophobic character and hydro-philic group. The hydrocarbon chasns associatewith each other to form micelle above a con-centration called c.m.c.** It may be expectedthat the hydrocarbon chains penetrate intothe hydrophobic fabric of a protein moleculeto enhance conformational change. The con-formational change of the protein moleculehas recently been investigated by the methodsof optical rotatory dispersion (//—15) andultraviolet absorption difference spectrum (16).

J i r g e n s o n s found that an addition ofdetergents to proteins having a value of ba

close to zero, e.g. ^-globulin, increased the valueof — b0 and that the reduced and carboxy-methylated albumin showed a conformationaldependent-Cotton effect in far ultraviolet region(11—14). These findings suggest that detergentfacilitates the formation of ordered structurewhich differs from that of the native molecule.While, it was found that, with increase in

* Present address ; Tezukayama University, Nara.•• The abbreviation used; c.m.c, the critical

micelle concentration.

the concentration of anionic detergent addedat pH 7, bovine plasma albumin showed anexpansion which was very similar to isomeri-zation (17) caused by increasing acidity (15).

The present investigation was undertakento obtain further information on the mecha-nism of the action of detergent on proteinconformation. The effects of three differentkinds of detergents, iur., anionic, cationic andnonionic detergents on the conformation oftwo different proteins were studied as func-tions of pH and detergent concentration. Oneof the proteins is bacterial a-amylase [EC 3.2. 1. 1, a-1, 4-glucan 4-glucanohydrolase. Bacil-lus subtilis), which has a folded structureincluding an a-helical structure withoutdisulfide linkage. The other is Bence-Jonesprotein which is found in the urine of patientsuffering from multiple myeloma. This proteinhas probably intrachain cross /S-structure butnot a-helical structure as observed for y-globulin (18). The effect of detergent on thedenatured a-amylase, in which a-helical struc-ture had been destroyed, was also examined.The experimental results showed that theoriginal conformation of the protein wasbroken completely and that the unfoldedpolypeptidc chain assumed to take partiallya-helical structure, content of which dependedupon the pH. Both anionic and cationicdetergents behaved as a helix-former even atextremely alkaline and acidic pH whilenonionic detergent did not have any helix-forming ability in the pH range of interest.

417

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418 A. IMANISHI, Y. MOMOTANI and T. ISEMURA

EXPERIMENTAL

Materials—Bacterial a-amylase was purchased fromNagase Sangyo Co., Ltd. (lot 20178) and purified bythe method described previously (19). Bence-Jonesprotein was kindly supplied by Dr. S. Migita. Thiispecimen was isolated from the urine of patient (Sh)with multiple myeloma by 1.8—2.5 M (NH4),SO4 andpurified by the procedure shown in a reference (20).A concentration of the protein (Sh) was determinedfrom absorption at 280m/< by assuming E]*,, =13.8*.This protein was found to possess II type antigeneticdeterminant on immunological assay (20).

Sodium dodecyl sulfate (SDS), anionic detergent,was purified and recrystallized from Emarl JO (lot549) given by the laboratory of Kao Soap Co., Ltd.The crude crystalline was recrystallized from 95 percent ethanol and washed with ethyl ether for removalof unreacted higher alcohol. The purified crystallinedodecyl pyridinium bromide (DPBr), cationic detergent,was purchased from Nippon Yuzai Kogyo Kai. Poly-ethylene glycol nonyl phcnyl ether, nonionic detergent,was given by the Research Institute of Fuji Film Co.,Ltd. The mean degree of polymerization of ethyleneglycol was 20.

Stock solutions of the proteins (approximately 2per cent) were made in 0.1 M NaCl solution. Eachsample solution used for optical rotatory dispersionmeasurement was made by mixing of 1 ml. of thestock solution with 1—5 ml. of buffer solution ofvarious pH's (0.1 M Tris, acetate and glycine buffer)and 0.2 ml. of detergent solution of various concen-trations. Sample solutions for ultraviolet absorptionmeasurements were made by further dilution of thesample solution for optical rotatory dispersion measure-ments with the buffer solution of the same pH. Themeasurements were carried out at room temperature20 hours after the preparation of measuring solutions.Concentration of detergent added to the protein wasexpressed by molar ratio of the detergent to theprotein.

Methods—A Rudolph photoelectric spectropolari-metcr model 200S was used for the measurement ofoptical rotatory dispersion. For the measurement ofan effect of temperature on the optical rotatory pro-perty a Perkin-Elmer photoelectric spectropolarimetermodel 141 was used. Temperature of the cell wascontrolled by circulating water of constant temperaturethrough the water jacket. The data were treatedaccording to the Moff i t t -Yang 's equation,

[«"'] =

100" n*+2

[a]--

S. Migita, private communication.

where [a] is specific rotation, [m1] the effective residuerotation, Mo the mean residue molecular weight, nthe refractive index of the solvent, a0 and b0 theconstants (optical rotatory dispersion parameters), and/i0 the constant which was taken as 212 mp, respectively.A change in the value of the Lorentz field correction(3/n'4-2) for the aqueous solvent due to the additionof detergent was quite small, i.e., it did not exceedmore than 4/1000 of the correction value for water.The correction for urea solution of various concentra-tion was made by the use of refractive indices reponedby S c h e l l m a n (21).

Ultraviolet absorption difference spectra weredetermined by using a Cary 14 MP automatic record-ing spectrophotometer from 250 to 320 mfi. The detailson the procedure of the measurement were describedpreviously (22).

Infrared spectra of the protein in D,O and inD|O containing SDS were obtained by using a Perkin-Elmer spectrophotometer Model 221 from 5 to 8fi at20°C. Protein concentration used was about 7 per cent.A KRS-5 cell with 0.05 mm. path was used. A CaF2

cell was us:d for the measurement with the solutionsat extremely acid pH.

Amylase activity was determined by F u w a'smethod using amylose as substrate (23^).

RESULTS

A. Bacterial a-Amylase

Spectral Shift Produced by the Addition ofSDS—Fig. 1 shows the difference spectra ofbacterial a-amylase in a buffer soultion ofpH indicated containing SDS referred to thesame protein solution of pH 7.2 without SDS.This spectral change considerably differes fromthose observed in the enzyme denatured byconcentrated urea or acid; a broad shoulderaround 295 raft and minima at 287 and 280 m^are distinctive. The spectral change dependedon the incubation period. After 20 hours nofurther change was observed.

The Effect of Concentration of the Detergent onDifference Spectrum, Optical Rotatory Propertiesand Enzymatic Activity—Fig. 2 shows the effectsof the anionic detergent (SDS) on the opticalrotatory properties, difference spectrum andenzymatic activity of the enzyme as a functionof molar ratio of the detergent added to theenzyme at pH 6.3. The enzyme has a negativenet charge at pH 6.3. No precipitation oc-cured by adding SDS in the range of molar

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Interaction of Detergents with Proteins 419

ratio indicated. Enzymatic activity decreasedwith an increase in molar ratio of detergentto the enzyme, and the remaining activitywas found to be ca. 20 per cent at a molar ratioaround 700. The activity measurement of theenzyme was made at the concentration whichwas 10* fold lower than that used for opticalrotatory dispersion measurement. Recoveryof the enzymatic activity could not be observedso far as the enzyme solution was dilutedwith a buffer solution without varying themolar ratio of SDS to the enzyme. A blueshift increased with increase in the molar ratioratio of SDS to the enzyme. At a molar ratioof 1,500 the molar extinction of a minimumat 287 mp was found to be 8,000. The valuewas about 40 per cent of that observed forthe enzyme denatured by 8M urea. Thevalue of b0 changed scarcely in the range ofthe molar ratio of SDS to the enzyme studiedwhile the value of a0 changed. This meanssome conformational alteration of the moleculeoccurs.

270 260 290

WAVELENGTH (

300

FIG. 1. Difference spectra of bacterial a-amylase.

Curve 1 (—•—), the urea-denatured enzymein 5.7 M urea (pH 1.3) vs. the native one (pH 7.2);curve 2 (—•—) , the acid-denatured enzyme (pH1.6) vs. the native one (pH7.2); curve 3 (—O—),4 (—•—) and 5 (—X—), the SDS-treated enzymeat pH6.3, 5.7 and 7.7 (the molar ratio of SDS tothe enzyme, 1,500) vs. the native one (pH7.2),respectively.

Fio. 2. Dependence of the optical rotatorydispersion parameters, difference spectrum (Je at287 m/j) and enzyme activity of the enzyme onthe concentration of SDS added at pH6.3 (0.1 MTris-buffer solution).

The Effect qfpH on the Interaction of Detergentwith the Enzyme—The interaction of detergentwith an enzyme molecule was affected by thepH of solution, that is, by the net charge ofthe enzyme molecule. Fig. 3 shows the pH

8 2PH

Fio. 3. The pH dependence of optical rota-tory dispersion parameters (left side of the figure)and difference spectra of the enzyme and theSDS-treated one (right side). The molar ratio ofthe detergent added to the enzyme is 1,500.Open and filled circles represent the value of a0

and b0 in the presence of SDS, and open andfilled squares those in the absence of SDS. Opentriangles represent the molar extinction coefficientof minimum at 290 raft (4e) in the differencespectrum of the enzyme at pH indicated referringto that at pH6.3. Filled triangles, • and T,represent Je of minima at 287 m/i and 280 m/j inthe difference spectrum of the SDS-treated enzymeat pH indicated referring to the native enzymeat pH6.3.

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420 A. IMAKISHI, Y. MOMOTANI and T. ISEMURA

dependence of the optical rotatory parameters,a0 and bg, and spectral change of the enzymein the presence and absence of SDS anions.Optical rotatory parameters of the nativeenzyme were found to be —130° and — 160°for a0 and b0, respectively (pH 6—10) (22).In the pH range from 5.4 to 4.0 optical rota-tion of the enzyme could not be measuredbecause of an increase in turbidity of thesolution. Below pH 4 the value of negativea0 began to increase accompanying simul-taneous decrease in the negative b0 withincreasing acidity. At pH2.8 the values of—441° and —75° were observed for those a0

and b0, respectively. In ultraviolet absorptionmeasurement a steep shift to shorter wavelengthof the spectrum of the enzyme was observedbelow pH5 (22). On the addition of SDSthe negative a0 decreased and blue shift indifference spectrum increased with increasingacidity of the solution while the negative b0

scarcely varied in the pH region above 5.5.Below pH 5.4 the negative a0 showed a tendencyto restore the original value and furtherspectral change was not observed except smallvariation of a peak at 280 m^ in the presenceof SDS. It is significant that in this pHregion the negative value of b0 (-150° to-160°in the native state) increases with increasingacidity in contrast with the decrease in b0 inthe absence of SDS. It seems that the molecularconformation below pH5.5 is presumably dif-ferent from that in the above pH region.

The Effect of SDS on the Urea DenaturedEnzyme in the Presence of Concentrated Urea—Native bacterial a-amylase has a value of b0

of —160°. In order to investigate the abilityor SDS to increase the negative ba of theenzyme, SDS was added to the solution of theenzyme which had been unfolded completelyby acidified urea solution. The values of 60

and Oo of the denatured amylase by acidic8 M urea solution in the absence of SDS werefound to —20° and —500°, respectively.

As shown in Fig. 3, at extremely low pHthe addition of SDS caused an increase inthe value of — ba accompanying a decrease inthat of —ao. When SDS was added to thedenatured amylase in acidic urea solution, a

negative value of — b0 was also obtained asobserved for ovalbumin by M e y e r andK a u z m a n n (23). Fig. 4 shown the depend-ence of the parameters and difference spectrumon the concentration of SDS added to theunfolded enzyme in the acidic 8 M urea solu-tion. Difference spectra of the urea denaturedenzyme produced by adding SDS in variousmolar ratio to the enzyme were measured byreferring to the same solution of the enzymein the absence of SDS. The higher molarratio of SDS to the enzyme increased, thelarger red shift of the spectrum and the greaternegative value of b0 were observed. Fig. 5shows the effect of concentration of urea on theoptical rotatory dispersion parameters at aconstant molar ratio (900—1,000) of SDS tothe enzyme. When the concentration of ureawas increased, an increase in — a0 accompaniedwith a decrease in — b0 was observed. Thecurves of the parameters are similar to thoseobserved for the denaturation of protein. Thepresence of concentrated urea gave rise tomore or less a decrease of the effect of SDSon the protein structure.

B. Bence-Jones Protein

The Effect of Concentration of the Anionic',

-240-

3 10 ZOO 500[SOSl/(PROTEIN] (ndei/aoU)

Fio. 4. Plots of the optical rotatory dispersionparameters and of it at 291 mfi in the differencespectrum of the enzyme in 8 M urea solution ofpH 2.4 as a function of concentration of the deter-gent added. As at 291 mfi is a measure of redshift of the spectrum of the enzyme in 8Af ureasolution produced by addition of SDS at pH 2.4.

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Interaction of Detergents with Proteins 421

-200

i--280

-120 -

0 2 4 6 8CONCENTRATION OF UREA ( M )

Fio. 5. The effect of concentration of ureaon the parameters of optical rotatory dispersion ofthe acid denatured enzyme in a glycine buffersolution of pH2.4 containing SDS of 900 to 1,000of the molar ratio to the enzyme.

Detergent—Fig. 6 shows the effect of SDS con-centration on optical rotatory properties anddifference spectrum of the protein at pH7.4.An increase in the negative value of b0 andan increase in that of a0 were obtained withincreasing a molar ratio of SDS to the protein.Difference spectrum of the protein solution inthe presence of SDS referring to the nativeone showed a blue shift (Fig. 7, curve 3).The spectrum roughly resembled that observedfor bacterial a-amylase below pH5.7 (Fig. 1).

Effect of pH on the Interaction of SDS withBence-Jones Protein—Fig. 8 shows the p Hdependence of the parameters, a0 and bQ, ofBence-Jones protein in the presence andabsence of SDS anions in the solution. Thevalue of b0 for the native protein was foundto be approximately zero throughout the pHrange examined. When pH was lowered, thenegative value of a0 began to increase at pH4.5 and reached to 630° at pH 1.4. On theaddition of SDS to the protein at the molarratio of 240 at neutral pH the negative valueof ba increased to 120°—130° accompanyingwith an increase in that of aa from 300°to 500°. When the pH was lowered in the

10 50 COCSDSl/CPROTEIN]

Fio. 6. Plots of the parameters of opticalrotatory dispersion and of At at 294 m/j in thedifference spectrum of Bence-Jones protein solutionas a function of concentration of SDS added tothe protein at pH7.4 (0.1 M Tris-buffer solution).

presence of SDS, the value of b0 became morenegative and that of a0 varied simultaneouslyto the opposite direction; the negative valueof a0 decreased from 500° to 400°. In extremelyalkaline pH region, where the protein has thesame sign of net charge that of SDS, an in-crease in — b0 of Bence-Jones protein was alsoobserved.

Spectral Change Induced by Addition of theDetergent—Difference spectrum, shown in Fig.7 (curve 3), is considerably different in theshape and wavelength of the peaks from thoseproduced by ordinary blue shift as observedfor the denatured protein. For example, anegative peak at 291.5 ran for the aciddenatured protein may be assigned to trypto-phyl residue while the corresponding peak forthe SDS-treated protein appeared at a longerwave length (295—6 m/*). This spectrum wasalmost independent of pH throughout theregion studied. When the spectrum of theacid denatured Bence-Jones protein in thepresence of SDS was compared with that ofthe same protein in the absence of SDS, ared shift was observed (curve 2). It revealsthat SDS anions presumably bind to theunfolded protein molecule by electrostaticinteraction and hydrophobic forces, and form

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422 A. IMANISHI, Y. MOMOTANI and T. ISEMURA

7.5 -

5.0 -

2.5 -

-2.5 -

-SO -

-7.5 -

2S0

WAVELENGTH(mp)

Fio. 7. Difference spectra of Bence-Jones protein.Curve 1 (—•—): difference spectrum of the acid denatured protein at pH 1.4 (0.1 M glycine buffer soln.)

referred to the native one at pH7.4 (0.1 M Tris-buffer).Curve 2 (—O—): difference spectrum of the acid denatured protein in the presence of SDS of the molar

ratio of 354 at pH 1.4 referred to the spectrum of the same protein at pH 1.4. (A redshift of spectrum of the denatured protein is produced by adding SDS.)

Curve 3 (—X •): difference spectrum of the SDS-treated protein at the molar ratio of SDS to the proteinof 354 at pH 7.4 referred to the spectrum of the native protein at pH 7.4. (A blue shiftof spectrum of the native protein is produced by adding SDS.)

a hydrophobic fabric around the molecule.Therefore, the difference spectra, as shown inFig. 7 (curve 3), suggest a difference betweenthe medium around chromophoric residues inthe native conformation and that in thehydrophobic fabric produced by associationof SDS anions.

Infrared Spectra of Bence-Jones Protein inDt0 in the Presence and Absence of SDS Anions—Infrared spectra observed for Bence-Jonesprotein in the sodium chloride region areshown in Fig. 9. It was seen that amide IIband, which was originally located at 1550cm."1, shifted to 1450 cm."1 upon deuterationof the NH group. Spectrum of the protein inD tO containing SDS at a molar ratio of SDSto the protein of 250 showed a shift of the

amide I band (at 1638 cm."1) to higher wavenumber accompanying a shift to lower wavenumber of amide II band in comparison withthat of the protein in D,O. Optical rotatoryparameters observed for the SDS-treated proteinused for the measurement of the spectrumwere found to be -505" and -139° for a0

and bo, respectively. Assuming that thenegative value of b0 is a measure of the con-tent of a-helical structure, the helical contentof the protein was estimated as about 25 percent. Fig. 10 shows amide I band for theacid denatured protein, the SDS-treated proteinat pD 1.2 and the native one. A band at1640 cm."1 observed for the native protein atpD 9.2 shifted to 1650 cm."1 by acidificationto pD 1.2. The band at 1655 cm."1 was

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Interaction of Detergents with Proteins 423

1 I 1 1 1 1 1 1 1—/—h [-200

Fio. 8. The pH dependence of parametersof optical rotatory dispersion of Bence-Jonesprotein and of the SDS-treated one. The molarratio of SDS added to the protein, 240—350.Open and filled squares represent the value ofa,) and b0 in the absence of detergent, and openand filled circles those in the presence of deter-gent, respectively.

1.800 I.TOO 1600 1500WAVE NUMBER (cm."1)

1400

Fio. 9. Infrared spectra of the Bence-Jonesprotein in D,O in the presence ( ) and theabsence ( ) of SDS. The molar ratio of SDSadded to the protein; 275, pD 7.0.

•observed upon addition of SDS to the proteinat pD 1.2. Optical rotatory parameters observedon this solution were found to be —371° fora0 and —161° for b0.

The Effects of Cationic and Nonionic Deter-gents on the Optical Rotatory Parameters of Bence-Jones Protein—The values of parametersobserved at various pH are summarized inTable I. It can be seen that cationic deter-gent also has an ability to increase a negativeJ>0 as suggested by M e y e r and K a u z m a n n

IOOr

80

60 -100-

- eo -

I.TOO 1,650 1,600

WAVE NUMBER

I.55O

Fio. 10. Infrared specta of Bence-Jonesprotein in three different states, ® ; the nativeBence-Jones protein in D,O (pD9.2), @; the aciddenatured protein in D.O (pD 1.2), (J); the SDS-treated protein in acidified D,O (pD 1.2), themolar ratio of SDS to the protein; 440.

TABLE I

The Effect of Cationic and Nonionic Detergents onOptical Rotatory Dispersion Parameters of Bence-

Jones Protein

pH

7.4

6.0

2.5

1.4

7. 1

1.4

-350°

-470

-578

-543

-285

-630

- 40°

- 50

— 75

-100

16

- 22

[Det]/[Prot]'>

366 Cationic Det.

// //

// //

// //

222 Nonionic Det.

// //

1) Molar ratio of detergent added to the protein.

for ovalbumin (73). Under the condition usedhere, the lower the pH value of the solutionbecame, the more negative value of b0 wasobtained. An increase in the negative a0

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424 A. IMANISHI, Y. MOMOTANI and T. ISEMURA

-300

"c

-350

- 4 0 0 -

-4O0

ac-450

-500

-450

Benca- Jones Protein

-I03O -190

-100

pH7.4MR-I25O-

_J I I I 1 I U20 30 40 50 60 70

TEMPERATURECC)

_J I 1

-too

4,

-50

-130

-SO

80 90

Fio. 11. The optical rotatory dispersion para-meters as a function of temperature on the SDS-treated Bence -Jones protein. MR: the molarratio of SDS to the protein.

-150-

-200-

-250-

a-Amylaw1

pHMR

1.4-880 I

-ISO

-200

-250

-300

: — : - - - -4

: "" ^--__:

t i i i i i

pH 7.4MR-440

I . I . I I

- -200

- - I 50

-1-200

--ISO

- -50

20 so go40 50 60 70TEUPEflATURECC)

Fio. 12. The optical rotatory dispersion para-meters as a function of temperature on the SDS-treated bacterial a-amylase. MR: the molar ratioof SDS to the enxyme. Dashed line representsthe values obtained by lowering temperature.

suggests partial unfolding of the original con-formation. Neither the native nor the dena-tured conformation of the protein was affectedby the nonionic detergent. Details of theeffect of cationic and nonionic detergent willbe reported later.

Thermal Dependence of Optical RotatoryDispersion Parameters of Detergent-treated Proteins—Figs. 11 and 12 show the effect of temper-ture on the parameters of optical rotatorydispersion of detergent-treated proteins. Theparameters for Bence-Jones protein at pH7.4-were scarcely affected by raising the tempera-ture to 80°C although a small decrease innegative ba accompanying with an increase innegative a0 was observed at pH1.4. Thetemperature dependence of the parametersfor bacterial a-amylase at pH1.4 was similarto that for Bence-Jones protein at pH1.4.The thermal effect on the parameters for bothproteins was reversible and showed no trans-ition point. A steep increase in the negative a<>by raising the temperature occured irreversiblyin the SDS-treated amylase at pH7.4 ac-companying a decrease in the negative bQ.This result suggests that the enzyme moleculestill retains some original conformation.

DISCUSSION

An increase in negative value of a0 anda blue shift of spectrum of the proteins causedby addition of SDS suggest an unfolding ofthe native conformation. A significant featureof SDS with respect to the interaction with,protein was an ability to increase the negativevalue of b0 as indicated by J i r g e n s o n s(//). In order to analyse the conformationalchange of the proteins due to the addition ofdetergents, the observed values of a0 wereplotted against those of b0 on a set of differentdispersion measurements as shown in Fig. 13.Both of these values are related to each otherby the following relation,

if the parameters do not vary in the set ofmeasurements. This relation was first proposedby L e o n a r d and F o s t e r to test the in-variance of the helical parameters (a"~D and

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Interaction of Detergents with Proteins 425

i")* and mean intrinsic residue rotation coef-ficient (5*)* on serum albumin (17). Thevalue of />" is about —630° which is charac-teristic of the isolated a-helix. The value ofa"~D is assumed to be +650° for helical water-soluble ionic polypeptides at ionic strength0.2 (24). However, it varies considerably withsolvent. When afi" was plotted against Ajf" forthe denatured Bence-Jones protein and thosefor the detergent-treated one in the molarratio of the detergent to the protein above200, almost a linear relation was obtained.Slope of the straight line, that is, the ratioof a$~D to b$, was found to be about —1.25.Assuming b^— — 630°, a"~D and flj werefound to be +790° and -640° from the line,respectively. Similar plot was performed forbacterial a-amylase which is shown in Fig. 14.Bacterial a-amylase was not readily affected

-zoo

-300 -

-400 -

-500 -

-800 -

- 7 0 0- 50 -ISO -ZOO-100

ifFio. 13. Plots of values of a^ against those

of Ao on a set of different dispersion measurementsfor Bence-Jones protein in the absence and presenceof detergents. Filled circles ( • ) represent thedata for the native protein and half filled circles( 3 ) those for the protein denatured by acid oralkali. Open circles (O) represent the data forthe protein in the presence of SDS of variousmolar ratio to the protein, open squares (O) thedata in the presence of DPBr and open triangles(A) the data in the presence of nonionic detergent.

• If the protein consists of an cr-helical part anda disordered part, Mof f i t t parameters are relatedto the helical content as follows;

obi _R , H - D , ,obi , ,H

<»0 = a o + / H "o and An = / H AQ

where fa is the fraction of a-helix (24 ) .

-100-

-zoo -

-300 -

-500 -

-600-150-100

A *

FIG. 14. Plots of values of a^ against thoseof Ao on a set of different dispersion measurementsfor bacterial a-amylase. g*Sl represents the rangeof the value for the native amylase. Half filledcircles (3 ) represent the data for the urea de-natured amylase in acid and neutral pH region.Circles (8 ) represent the data for amylase in con-centrated urea in the presence of SDS of variousmolar ratio to the enzyme, open circles (O) thedata observed for amylase below pH5.7 and thosefor the acid denatured-neutralized amylase inthe presence of SDS and squares (Q) data for theamylase in the pH region below 6.3 in the presenceof cationic detergent (DPBr). Crosses (X) re-present the data for amylase at pH 6.3 in thepresence of SDS of the molar ratio below 290 andthose for the enzyme in the pH region between7 and 10 in the presence of SDS of various molarratio to the enzyme.

by adding SDS in the pH region from 7 to9 where the molecule has a quite stable con-formation in aqueous solution. These dataare represented by crosses in Fig. 14. A linearrelation is obtained if the observed values ofaa for the. enzyme solution of various pH'sbelow 6.3 containing detergent in variousmolar ratio to the enzyme above 290 areplotted against those of b0 in the presence andabsence of the concentrated urea. The slopewas found to be —1.1 and assuming thevalue of -630° for b", the values of +690°and —400° were obtained for a j 1 " 0 and aj ,respectively. There is a slight differencebetween the slope, the ratio a£~D/b", obtainedfor Bence-Jones protein and that for bacteriala-amylase. It has been reported that the

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426 A. IMANISHI, Y. MOMOTANI and T. ISEMURA

value of a" D considerably depends not onlyon the solvent environment^ 24) but also onthe nature of the side chain groups on poly-peptide chain (25) while the value of 6" issubstantially the same for various polypeptidesin various solvents as helix former (24).Therefore, it may be likely that the value ofa^~D, which reflects the nature of side chaingroups involved in a-helical region in a proteinmolecule, slightly varies from one protein toanother one. In view of the facts describedabove, it may be inferred that a linear increasein negative b0 with a decrease in negative a0

caused by addition of the ionic detergent ismainly due to the development of a-helicalstructure providing that the value of /Jo isalmost a constant throughout the pH rangeexamined either in the presence or in theabsence of detergent Under the above con-ditions both these proteins lose their originalconformations and assume to have differentconformations, including a partial a-helicalstructure, from that in both the native andthe denatured states, although it can not bededuced for bacterial a-amylase whether ornot a part of the peptide chain assuming a-helix in the detergent-treated state is the samewith a helical part in the native state. Thisinference is confirmed by the thermal depend-ence of the parameters for bacterial a-amylaseat two different molar ratios of SDS to theenzyme as shown in Fig. 12: The thermaldependence observed for the enzyme at MR880 at pH1.4 (taken as an example of thedata represented by open circles in Fig. 14) wassmall and reversible while that for the enzymeat MR 440 at pH 7.4 (croses in Fig. 14) showedan irreversible transition. This transition sug-gests that the original conformation stillremains in the latter case. The negative valueof a* (400°) deduced for bacterial a-amylase isconsiderably smaller dian that for Bence-Jonesprotein, althought this cannot be expectedfrom the larger negative value of af* (500°)for the enzyme in urea denatured state. Atthe present stage, it cannot be explained, buta tentative explanation is that the addition ofsuch a small amount of detergent that thenegative value of b0 can be scarcely produced

causes a change of tertiary structure of themolecule which affects positively on the valueof a0 alone.

The data shown in Fig. 13 can account forthe phenomenon observed by J i r g e n s o n s(14) that both specific rotation and b0 werechanged to more negative values on the addi-tion of detergent to the native protein : Forexample, when SDS was added to Bence-Jones protein in the molar ratio below 200,SDS anions gave rise only to a partial unfold-ing of the native conformation accompanyingwith simultaneous refolding of a part of theunfolded polypeptide chain to a-helical struc-ture. This transconformation increases anegative value of b0 and results in furtherincrease in negative value of a0, i.e., thespecific levorotation. The negative a0 valuewas, however, cancelled partially by thepositive contribution due to the developed a-helical structure. In the condition that thenegative value of b0 reached to the maximum,the negative value of aQ was found to bestill larger than that for the native protein asshown in Fig. 13.

Nonionic detergent seems to have no effectto increase the helical content of these proteins.In the cases of ionic detergents, cationic de-tergent as well as anionic detergent affects toincrease the helical content even if the proteinhas the same sign of net charge with thoseof the detergent ions. However, the effect ofcationic detergent is less than that of anionicdetergent: In bacterial a-amylase havingrelatively higher value of — bo, cationic deter-gent affects on the conformation to reducethe content of a-helix (squares in Fig. 14). Butfor the denatured enzyme or the protein withb0 close to zero the detergent restores or deve-lops a-helix even in the pH region where theprotein assumes to have positive net charges.On the addition of anionic detergent the deve-lopment of a-helix depends considerably onthe pH while the blue shift of the spectrumdoes not. These results suggest that the elec-trostatic interaction between ionic detergentand the oppositely charged groups on theprotein molecule plays a much important roleon the conformational alteration. A tentative

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Interaction of Detergents with Proteins 427

speculation on the interaction described in the following may be allowable : For the forma- tion of a-helix of polypeptide chain, the charg- ed groups on the chain must be neutralized a t first by electrostatic binding of anionic or cationic detergent ions. For example, in the interaction of SDS with protein, SDS anions bind to amino groups on the molecular chain a t the begining and increase a hydrophobicity in the neighbourhood of the binding sites. The remaining oppositely charged groups, that is, carboxylate ions, are protonated one by one with increasing acidity. This neutralization favors the formation of hydrophobic fabric due to the association of SDS around the peptide chain. While, the nonionized chromophoric residues may be imbeded independently of p H in the hydrophobic fabric produced by SDS ani- ons because of its strong hydrophobic character. In the hydrophobic fabric, polypeptide chain might be almost free from an effect of water, so that the a-helical structure should be developed or restored stably through intra- peptide hydrogen bonding which is more strengthened by the nonpolar medium ( 26).

A red shift of spectrum of Bence-Jones protein as shown in Fig. 7 (curve 2) reveals that the chromophoric residues are surrounded by a hydrophobic fabric produced by associa- tion of SDS anions. A similar large shift to longer wavelength was observed for bacterial a-amylase when SDS was added to the enzyme in 8 M urea solution of p H 2.4 (Fig. 4). These shifts always accompanied with an increase in negative bo, i.e., an increase in helical content. The negative value of bo for the SDS-treated protein, however, decreased to about 60 to 70 per cent of that of the value in the absence of urea. This is the case that urea plays a role to weaken hydrophobic interaction (27) in such a manner that it decreases slightly the stability of detergent micelle in water (28). Addition of dioxane to the protein in the detergent solution also gave rise to an effect which was similar to that of urea (decreasing in the value of -bo and increas- ing in thatof -ao).* C o r r i n a n d H a r k i n s

* A. Imanishi, unpublished data.

has reported that dioxane increases slightly the critical micelle concentration of cationic detergent in water ( 2 9 ) . It has been also found that dioxane decreases the stability of hydrophobic region in protein molecule* while it contributes to stabilization of a-helical configuration of water soluble polypeptide

( 30). These results suggest that hydrophobic interaction between protein and detergent molecule is very important for the develo- pment of a-helical structure. I t was also found that the helical structure of proteins developed in the detergent solution was little affected by temperature up to 80°C. This may be also expected from the endothermic formation of the detergent micelle (31 ) if it is assumed that the helical part is surrounded by an hydrophobic atmosphere produced by

association of hydrocarbon chains of the detergent.

In comparison of Fig. 6 with Fig. 2, i t can be seen that Bence-Jones protein is more easily affected by detergent than bacterial a-

amylase in the stable p H region. This dif- ference may be due to difference in charge distribution on the molecular surface between them : In the molecule of bacterial a-amylase, the oppositely charged groups display intra- molecularly a strong electrostatic interaction due to the pairing as expected from the abnormal behavior found in the p H titration ( 22 ). Therefore, the approach of detergent ion to the molecular surface of the protein should be reduced. While, it can be expected for Bence-Jones protein from the data on p H titration of 7-globulin (32 ) that the ionizable groups on the protein distribute evenly enough to behave normally in pH titration. Then, the electrostatic binding of detergent ion to the protein may be easier than to bacterial a-amylase.

E d e l m a n and G a l l y found that amino acid composition of Bence-Jones protein was identical with that of the L-chain of myeloma 7-globulin and that thermosolubility and spectrofluorometric behavior of the protein - --

* K. Hamaguchi, private communication.

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428 A. IMANISHI, Y. MOMOTANI and T. ISEMURA

and isolated L-chain were also similar (33).This protein (34) as well as /-globulin (35)and myeloma protein (36) has a value of b0

approximately zero. Recently, I m a h o r i h a sfound the existence of intramolecular fi-structure in /-globulin fibril by infrared spec-troscopy (18). Intrachain cross /3-structureshows a value of b0 of approximately zero(37) while interchain parallel /3-structure hasa positive value which depends on the con-centration of polypeptide (38). For Bence-Jones protein the existence of /3-structure canbe expected from its similar physical andchemical properties to those of /-globulin. Inthe infrared spectrum, it has been found thatamide I band for the film of polypeptidechain having cross /3-structure appears around1637 cm."1 (39) while it shifts to lowerwavenumber, 1620—10 cm."1, for the deuteratedone having same structure (40). This uniquefrequency has not been observed for /-globulin(35, 36) and for myeloma protein (36) indeuterium oxide. Infrared spectrum of Bence-Jones protein in deuterium oxide showed theamide I band at 1638—40 cm."1 When SDSwas added, the amide I band shifted to1650 cm."1 Similar shift in wavenumber ofthis band was also observed by acidificationto pD 1.2. The band around 1650 cm."1 isdue either to a disordered structure or to ana-helical structure. The observed band at1638—40 cm."1 for the protein at pD 7.0 isidentical with that (1637 cm."1) for the film ofpolypeptide having cross ^-structure. Thepossible existence of /3-structure in the proteincannot be deduced from this frequency alonebecause deuteration of amide groups is con-siderable under the experimental condition.However, infrared spectrum of deuteratedlysozyme [EC 3.2.1.17], which has beenestablished to have /S-structure as well as oc-hclical structure, shows amide I band at1630 cm."1, which is considerably higher thanthe value for deuterated polypeptides having/9-structure, besides at 1652cm."1 (41). Thelower wavenumber of amide I band for Bence-Jones protein at pD 7.0 and the shift to higherwavenumber by SDS-treatment or acidificationmay suggest that there are some parts of poly-

peptide chain assumed /3-structure in themolecule at pD 7.0 and that they are rupturedby the treatment*.

SUMMARY

The effects of three different kinds of deter-gents, namely, anionic, cationic and nonionicdetergents, on the conformation of two differentproteins were studied by the measurements ofoptical rotatory disperson and ultravioletspectra and infrared spectra as functions ofpH and concentration of the detergents. Oneof the protein examined is bacterial a-amylasewhich has a folded structure of single poly-peptide chain without disulfide linkage. Theother one is Bence-Jones protein which maycontain an intramolecular /3-structure but notan a-helix. The experimental results revealedthat change in optical rotatory dispersion byadding detergent was due to destruction ofthe original conformation followed by a part-ial a-helix formation of the unfolded poly-peptide chain, and that the extent of thea-helix formed depends upon the pH value.This refolded polypeptide chain was imbededin a hydrophobic fabric produced by deter-gent associated around the polypeptide. An-ionic and cationic detergents act as a helixformer even at extremely alkaline or acid pHregion. On the other hand, nonionic detergenthas not such an action throughout the pHregion studied.

The authors wish to thank Dr. K. Hamaguchifor his helpfull discussions during this work, Dr. K.Fukushima for his valuable advices on the interpreta-tion of infrared spectral data and also Mr. H. Matsuurafor the measurements of infrared spectra. The authorsare also indebted to Dr. S. Migita for the generousgift of Bence-Jones protein which made this workpossible and to Mr. H. Tokiwa (Kao Soap Co., Ltd.)for the gifts of detergents.

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* This consideration has been supported by thespectrum of the film of Bence-Jones protein in theregion 600—800cm."1 which shows the existence of^-structure; K. Fukushima, unpublished data.

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Interaction of Detergents with Proteins 429

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