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Isoelectric points of proteins, determined by isoelectric focusing in the presence of urea and ethanol
CElROM.
.”
11,550
ISOELECTRIC POINTS OF PRO-@INS, DETER&NED FOCUSING IN THE PRESENCE‘QF UREA AND ETH~OL
.. .
W. J. GELSEMA,
C. L. DE LIGNY
and N. G. VAN
DEEC VE&
Analyiica Ciiern~Oy hboralory. Sfate UnmerSfy Wt~eciit,Croesstm&~~tz, &ree&&(The Nerhcr; [&.I (Fust received May lOth, 19s; revised manuscript re&ved September 29$1918)
.I ‘. . SUMMAFW
..
-_
-_ ‘.
rsoetectric points, plep9, in sufxose-urea-wter and gfycerokhano@i+% mixtures and isoekctric points, PI, in water have been dete~ed at 29 foriscarrier ampholytes. ‘T&edifferences,pf,, -pI, are shown to account fqr the prnqaq mediumeffkctand the pH measuringcelfeEectonthe i&ekctric‘point~Thedifiken~~s, protf&s. _ Pl,,, -pi, for Ampholiks are used to correct apparentisoektric points pl shifts resulting from the denaturing effket of urea and et&&31 are diQ+sed in terms of the conformation change_
of
INTRODUCTiON In a recent series ofpapersf3, we showed that the apparent isoekctri~ #z&s, of proteins, measured by isoelectric focusing in densiw gradients of SUCRIS% .$&ol or ethy!ene glycol in the presenceof mrrier amph&tes, can irec&e&d fk the primary medium effect and the pH mezxsuring-CeEf effect tg giv&i&kAri~ points;pi, in water. The correctionterms to&? used, yhich wereta+~Iate@~,depend 06 p&,&i the concentration of l he non-electrolyte, the tempetatirre&d,the &emicaE nature of the carrierampholyte, fn this approach, it was tacitly assUmedthat t& c~nforni&m ,. of proteins is not affected by the pdient-forming non&x-tro~ytL T&e is; indeed, enough evideke4~’ in favour of this assumption with sacrose, -g&&of-and ethylene gIyco1at the concentrations used in isoekctric fkxising. In a paper dealing with the effect of urea on the behavitiurof so&e pro&ins in ’ isoelectricfocusing, UP demonstratedthe value of measurementsof isoelectricpoin& in the absence and presenceof urea for conformationaEstudiesof proteink-However, : ’ in that paper, isoekctric points measured in the presenceof u&k we& ?Q& foi the primarymedium effect and the pH measuringcell effect due to t&d?.& aqekge correction term, independent of p&,1 In- view of the .evidenceres&i~~g.f?om ow wodP, this procedure appears to be iiicorrectl. Moreover, literates W&ES ofax-. rection terms, pl,,, -pI, associated with the effkd of WC& k&e a. cotid+a$Ie : i scatter,as is shown in Table I. As we agree with W on the V&I& of isue!ectk&using for conformatikkaf
W. J.
172
TABLE I LITEE+4TURE VALUES OF PI.,, Urea concrntration (Ml
Correction PL -PI
6 6 7 7
0.42 0.3; 0.4 0.9 0.35
-
GELSEMA, C. L. DE LIGNY, N. G. VAN DER VEEN
pZ
term,
USED TO CORRECX FOR THE EFFECT OF UREA Range of PI.,, for which correction term is stated to be valid
Reference
4-10
4-6; 7-10 3-6 2.5-6
8 9
studies of proteins, we determined the appropriate correction terms accurately. For p1 for Ampholines in the presence of two this purpose we measured values of PI,,,reagents that have a conformation-changing effect on proteins, viz., urea and ethanol*, as a function of the concentration of these reagents and of the PI=,,, values of the Ampholines. As the effect of urea or ethanol on the conformation of proteins can be studied by density gradient isoelectric focusing, these PI,,,-plvalues were determined also as a function of the concentration of two gradient-forming non-electrolytes, viz., sucrose and glycerol. Supposing that urea and ethanol do not affect the conformation of Ampholines, these values of PI,,, -pl can be assumed to account correctly for the primary medium effect and the pH measuring cell effect of the reagents on the isoelectric point of a pr&ein. Any difference between the isoelectric point of a protein in water and that obtained by correction of the pl,,, value measured in the presence of the conformation-cha’nging reagent can then be associated with the conformation change of the protein. It should be borne in mind, however, as was pointed out by UP, that the absence of a pl shift due to a conformation-changing reagent cannot always be interpreted in terms of retention of the native configuration, as the pl value is informative only for the degree of dissociation of a few protolytic groups. As a test of the validity of the correction procedure, we reappraised the literature values of isoelectric points of some proteins in the absence and presence of urea and measured the isoelectric points of two proteins, viz., ribonuclease and p-lactoglobulin, in the absence and presence of urea and ethanol. In those instances where a significant shift in corrected isoelectric point was found, this shift was interpreted in terms of the conformation change by comparing the result with calculations on the basis of the equation of Linderstmm-Lang and Nielsen’“, using the known chemical compositions of the proteins in question.
While urea and ethanol are both frequently used in conformational studies of proteiz”, cnly the former has been used as such in conjunction with isoelectric focusing. There is. however, no major objection to the use of ethanol in isoelectric focusing for conformaticnal stcdies. Practical drawbacks are, of course, its low density, which induces a lower stability of glycerol density gradients. and its high volatility, which can be expected to render isoelectric focusing experiments cn granulated gels more dificult. To our knowledge, such gel-isoelectric focusing experiments hale net teen carried out; experiments in a glycerol gradient, containing up to 60% of ethanol, have heen Ferfcrmed by Letedev et al.” for the separation of water-insoluble ccm proteins. l
i.WELECTRX
PQXNTS OF PROTEINS
173
EXPERIMENTAL
Measurements of isoelectric points of ‘Carrier ampholytes
The isoelectric points of 2 % (w/v) solutions of Amphoiines (LKB, Stockholm, Sweden) and Servaiyte (Serva, Heidelberg, G.F.R.) in water (pr) and in the presence of urea, ethanol, urea + sucrose and ethanol f glycerol (p1,,,) were determined at 25” as described eariieti. The chemicals used were urea (Merck, Darmstadt, G.F.R., Cat. No. 8488), ethanol (Baker, Phillipsburg, N-J., U.S.A., anaiysed grade), sucrose (Baker, analysed grade) and glycerol (Merck, p-a. grade). The specific conductivity of an aqueous 6 M solution of urea was 9.7 - 10-6R-s.cm-1 at 25”, indicating the absence of eiectrolyites. Urea was dissolved just before the measurements without raising the temperature above 25”, to prevent the formation of cyanate’“. At relatively high concentrations of ethanol the reduced soiubiiity of the carrier amphoiytes eesulted in turbid solutions. Measurements of isoelectric points of proteins
Aliquots of 15 mg of bovine pancreatic ribonuclease (Sigma, St. Louis, MO., U.S.A., Cat. No. R-4875) OF bovine B-iactogiobuiin (Serva, Cat. No. 27440) were focused in au elect&focusing coiuruu (LKB 8100-l) ‘at a temperature of the cooling water of 4”. The concentration of Amphoiines (pH range 9-l 1 for ribonuclease aud 3.5-10 for @-iactogiobuiin) was 2% (w/v). Density gradients were produced with a gradient mixer (LKB 8121) and ranged from 50 to 5 % (w/w) of sucrose in the absence and from 42 to 4% (w/w) of sucrose in the presence of 6 M urea, and from 60 to 6 % (w/w) of glycerol in the absence and presence of 30 % (w/w) of ethanol. The cathoiyte was a 0.25 M sodium hydroxide solution; the anolyte was 0.16 M orthophosphoric acid OF 0.01 M acetic acid. After focusing at constant power (5 W) for 48 h (LKB 2103 power supply), the contents of the column were collected in fractions of 3 ml for ribonuclease and 1.5 ml for ,8-iactogiobuiin. The extinction at 280 nm of these fractions was measured using a Vitatron Type MPS spectrophotometer. Z&z pH at 25” of the fractions with maximum UV extinction was measured, and the sucrose OF glycerol content was determined from the refractive index. We used calibration graphs established with solutions of 2% (w/v) Ampholines and 6 &1 urea in sucrose-water mixtures of varying sucrose content and with solutions of 2 oA(w/v) Ampholines and 30 oA(w/w) ethanol in glycerol-water mixtures with varying glycerol contents, respectively. RESULTS
Isoelectric points of carrier ampldytes The results are given in Table II. As an example, Figs. 1 and 2 give plots of PI,,* -pl versus PI,,, at a constant concentration (6 M) of urea in sucrkc-water
mixtures and at a constant concentration (30 %, w/w) of ethanol in glycerol-water mixtures, respectively. Figs_ 3 and 4 show values of 1p,,,-p1 ~ers~.spf,,, in aqueous solutions of urea and ethanol, respectively. Isoelectric points of proteins
The results for pl,,,
at 25” are given in Table III. Table III also includes pl
RANGESANDFORSERVALYTEOFpHRANGE2-4
(%,1d11))
ngertt
.I
-
Glycerol(O%)(1.33) (20%)(1.75)
Ethanol (72%,w/w)
1
Glyccrol(O%)(0.75) (20%)(1.00) (40%)(1.38)
Ethanol (5O%,w/w)
_..
Glycerol(O%)0.38 (20%) 0.51 (40%) 0.71 (60%) 1.04
Ethanol (30%,w/w)
-.
Sucrose (0%) 0.85 (15%) 0.89
Urea(9M)
0.56 0.58 0.62 0.68
Sucrose(O%) (15%) (30%) (45%)
Urea(6M)
. ._. ._
(1.38) (1.75)
073 0,95 1.34
0.36 0.50 0.70 1.06
0.74 0.80
0.47 0.49 0.54 0.62
-_
-
0.62 0.81 I.13
0828 0,40 0.59 0.90
0.66 0.71
_ -
. -
(0.76) 0.87 __.._.. __.
0.33 0.45 0.65
0,09 0,17 0,29 0.47
0.52 OS5
0.33 0,34 0,37 0.42
0.16 0.15 0.16 3.19 0,23
0.20 O,l9 0.21 0.24 0.29 0.41 0.44 0.48 0.55
pfl(S-7) pl5.94
.~1
.__.,.._
.
.
(0.61) 0.60
0.30 0.38 0.48
0.11 0.17 0.25 0.34
OS2 0.56
0.33 0,35 038 0.45
0.16 0016 0,17 0.21 0.26
-
pli (6-8) ~16.90
.__._. _.___. .-._
pli(4-6) p14.87
,_.-.... -- --. .._..__ -_.._.._.. ___. .__..,__.
Antplfolitw
pH(2.5-4) pH(3,5-5) pI4.34 ~12.90 .---- --__-. ..._._ ..- .__._, ____.-.__.._..___-- .._- .-._. ._-._ Urea(3M) Sucrosc(O%) 0.27 0.21 0.21 (15%) 0.27 0.23 (30%) 0.27 0.26 (45%) 0.30 0.32 (55%) 0.33
cfmerrrrrttbtt
-.-.
-
-.
...-...
----
0.57 0.61
0.36 0.38 0.40 0.47
0.17 0,17 0,17 0.20 0.24
_.__
_._..
0.26 0.33 0.43
0.08 0.13 0.21 0.31
0.58 0.60
0.35 0.37 0.38 0.41
0.17 0.16 0.15 0.15 0.17
..---
-0.02 0 0.06
-0.08 -0.07 -0.05 0.01
0.50 0.49
0,31 0.30 0.26 0.24
0.15 0.11 0.07 0.02 0.01
--
.._
(1.13) (1.46)
(0.60) (0.83) (1.20)
0.26 0,38 0.56 0.89
0.80 0,84
0,53 0.55 0.58 O-66
;H(2-4) pl3.40 .._-.... ., 0.27 0.26 0026 0.2g 0,33
lyre
Servn-
__...,_
-_.
..
.
_.. .._ .. ..-........
.
p11(7-9) pN(8-9.5) pH(9-11) ~18.03 ~18.85 ~19.76
.I
was nradcina turbid solution. inpnrcnlhcscs dcnotcthatthecorresponding measurement of PI,,,,,, r&l - pf values ..-. ..-. ..---. .._.. _._.,__ _. .._ ._....__.._ ___: .._._ . ..__-- ,.. Ditratrrritrg GrflflierrrCirrier ~trrpholyte ._. ._. _. ..-._-_ . . ..- ._-.. ..-.. . -. ._ ._- ._. ngerit arrfl jiwtfrlll~
VALUES OF PI,,,, - pfAT25"FORAMPHOLINL?SOFVARlOUSpH
TABLE11
175
ISOELECTRIC POW-i-!3 OF PROTEINS
P&.)-PI
11: wo-
Ox)-
Fig. 1. Values of PI.,, - pl verse pl,,, for Ampholines (open symbols) and Servalyte (closed symbols) at 25” in 6 &l solutions of urea in water (a) and sucrose-water mixtures containing 15 (a), 30 (8) and 45% (w/w) (n) sucrose.
values at 25” calculated by applying correction terms, pz,, -p1, resulting from previous work3 and Table II in this paper. DISCUSSION
The results in Table II and Fig. 3 corroborate our prediction that the effect of urea on the apparent isoelectric point of an ampholyte depends on the acidity of the ampholyte. The same conclusion holds for ethanol (Fig. 4). Also in accordance with
120
.
too 0.80 / -
I
0.60 -
Phppjl
0.40 -
0200 _________
4201 3.
_ ____
4
5
_ _____
7
6
-_
_ __________
8
9
m-e_
10
PIwJ
Fig. 2. Values of PI.,, - pl versusPI.,, for Ampholines (open symbols) and Servalyte (closed symbols) at 25” in 30% (w/w) solutions of ethanol in water (0) and glycerol-water mixtures containing 20 (a), 40 W) and 60% (w/w) (A) plyceroi.
176
W. J. GELSEMA,
01
3-
4
5
6
C. L. DE LIGNY,
6
7
9
N. G. VAN DER VEEN
10
11
PbP
Fig. 3. Values of PI.,, - pl versus PI,,, for Amphohnes (open symbols) symbols) at 25” in aqueous solutions containing 3 (a), 6 (0) and 9 M (A)
and ServaIyte (closed urea.
PI,pp,-PI
-a20
j
4
5
6
7
8
9
10
%PD
Fig. 4. Values of PI,,, - pl versus pl.,, for Ampholines (open symbols) and Servalyte (closed symbols) at 25” in aqueous solutions containing 30 (S), 50 (o) and 72% (w/w) (A) ethanol.
TABLE
III
ISOELECXRIC
POINTS
OF PROTEINS
(25”)
Protein
Conformationchanging agent
&P
Gradient-forming agent (36, w/w)
PI,,,
Ribonuckase
-
9.50 9.53 9.Qo 9.75 9.81 9.82 4.98 4.99 5.40 5.38 5.76 5.74
38.6 % sucrose 40.7 % sucrose 27.5 y0 sucrose 46.5 % glycerol 48.6 % glycerol 47.8 % glycerol 34.2 y0 sucrose 34.8 % sucrose 28.0 % sucrose 28.2 0/0sucrose 49.3 % glycerol 47.3 % glycerol
-0.11 -0.12 0.28 -0.04 -0.05 -0.06 -0.04 -0.04 0.47 0.47 0.68 OX%
&Lactoglobulin
6Murea 30% (w/w) ethanol 30% (w/w) ethanol 30% (w/w) ethanol 6Murea 6Murea 30 % (w/w) ethanol 30 “/6 (w/w) ethanol
- pI
pl 9.61 9.65 9.62 9.79 9.87 9.88 5.02 5.03 4.93 4.91 5.08 5.10
ISOELECTRiC
POTN-iS OF PROTEINS
177
earlier findings3, the effects of urea f sucrose and ethanol + glycerol (see Figs. I and 2) on the pl,,, of Servalyte differ from those on the PI,,, of Ampholines of comparable acidity. A comparison of values of pZ*,, -pZ in sucrose-water and glycerol-water mixtures3with the results in Table II shows that the effects of sucrose and urea and of glycerol and ethanol, respectively, are not additive. As there are no literature data on the pK values of weak acids in ternary solvent systems, this finding cannot be commented upon. The general appearance of the curves in Figs. 1 and 2, which show the effect of sucrose and glycerol, respectively, is similar to that in Figs. 3 and 4, which show the effect of urea and ethanol, respectively, and to that in Fig. 1 in ref. 3, demonstrating the effect of ethylene glycol. This suggests that the pl,,, -pZdifferences due to the five mentioned non-electrolytes are caused by the same effects, viz., the primary medium effect and the pII measuring cell effect. As was pointed out in a preceding pape?, by combining values of pZ,,,-pZ with values of 6, accounting for the pH measuring cell effect, values of pZ’-plcan be calculated : pr-pZ
= pZ,,,-pZ--6
Subsequently it was shown in that pape$ that the pZ’-pZ values of Ampholines calculated in this way arc in between the pK’-pK values of carboxylic acids and alkylsubstituted ammonium ions, giving additional support to the view that the pZ,,,-pd differences of Ampholines due to sucrose, glycerol and ethylene glycol are indeed caused by the two effects mentioned above. 6 values due to ethanol are known lo_Therefore, pZ*-pZ values were calculated -0 5 with literature valuesis of pK’-pK. An analogous comand are compared in Ft,. parison for the effect of urea cannot be given, owing to the lack of corresponding 6 values. One can compare, however, (see -Fig. 6) the pl,,,-pZ values of Ampholines with literature values of pK,,, -pK for acetic acidI and the n-butylammonium ion.“. Figs. 5 and 6, which should be compared* with Fig. 3 in ref. 3, show that the effects of both urea and ethanol on the isoelectric points of Ampholines are indeed intermediate between the effects of these non-electrolytes on the dissociation constants of carboxylic acids and alkyl-substituted ammonium ions. We conclude, therefore, that the pZ,,,- pZ values in Table II can be used to correct for the primary medium effect and the pH measuring cell effect on the isoelectric points of proteins. Therefore, these values were used in a reappraisal of the literature values of the isoelectric points of some proteins, measured by isoelectric focusing in the presence of urea (see Table IV). The data, confined to proteins that are not dissociated into subunits by the action of urea, clearly indicate the importance of the correction procedure. For insulin and haemoglobin, there is no siguificant influence of urea on the pZ value, which leads to the conclusion that these proteins contain no abnormally dissociating groups, as far as groups having an influence on the isoelectric point are concerned. For a comment on this conclusion, which was also arrived at by Ui”, we -__ * For that reason,data on propionicacid and n-propyiamine are includedin Fig. 5.
W. J. GELSEMA,
178
C. L. DE LIGNY,
0
I
2
N. G. VAN DER VEEN
4
6 _a x; Molarityof lreo
Fig. 5. Values at 25” of pK* - pK for acetic acid (e), propionic acid (A), n-butylamm&ium ion (I) and n-propylammonium ion (V) and PI* - pl for Amph~lim~, pi = 2.90 (0) and p1= 9.76 (a), in ethanol-water mktures.
Fig. 6. Values at 25” of pk.,,
- pK for acetic acid (e) and n-butylammonium ion (a) and p&, and pZ = 9.76 (o), in aqueous urea solutions.
-
pZ for Ampholines, pZ = 2.90 (0)
TABLE
IV
LITERATURE OF UREA
VALUES
Protein
Urea concenfration
--.-
__--._____
OF ISOELECTRIC PL,,
WO)
l-M)
l
POINTS IN THE PRESENCE Concentration of sucrose f s, w/w) --
PI,,,
-
pl
AND ABSENCE
pI(2Y)
Reference
Insulin
0 6
5.69 6.15
20 20
-0.01 0.36
5.70 5.79
6 6
Haemogfobin
0 6
7.07 7.50
38 34
0.01 0.40
7.06 7.10
6 6
Ribonuclease
0 6
9.26 9.93
45 45
-0.08 0.26
9.34 9.67
6 6
a-Czsein
0 7
4.4”’ 4.66
-20
-0.8
4.4 3.9
8
0 7
4.5”’ 5.4
-20
-0.8
4.5 4.6
8
0
3.7; 4.1 ** 6.2
-20
-0.8
3.7; 4.1 5.4
8
/Kkein li-Casein
7
l
-
* PZ,~, (25”) was obtained from the literature value p&t) using~pl/ilTvalues equal to the mean of the distribution of dpK/dT values given in Fig. 2 in ref. 3. ** Estimated from the descriptions of the focusing experiments and the graphs of the focusing patterns. *** Moving boundary values.
ESOEFBXRIC
POINTS
OF
PROiEMS
-179
refer to his paper. For ribonuckas&, however, the significant shift in &due to .urea would imply the presence of abnormally dissociating groups in the native .stat$. The data on caseins show, contrary to the conclusion th& would be obtained Corn a comparison of p&,, values, that a-casein does and /kasein does not containanomalously dissociating groups determining the isoektric point in the native state_ This. is consistent with the evidence from viscosity measurements~*,indicating that ‘&case&r is a random coil in the native statti. :.:: In Table III, our experimental resdts on dboauckase are given. Whereas the value for pIestimated from the measurements ~FI 6 M urea agrees with thaffound.by UP, the p1 value found in the absence of urea differs _mnSiderabIy from his vafue (9.34). Our rest&s are in good agreement with the.pH of aiqueoussoiutions of rib& nuclease, found by Nozaki and ‘+SordLg (9-60 at zero.ionic strength and 9.71 in 0-U M potassium chloride solution) and by Tanford and IIauensteirP (9.65 in O;LS M. potassium chloride sohttion). We conclude therefore, in contrast to -UP,. that .6 A& urea does not produce a signScant pi shift_ Further, there is no need for he assump tion ofan abnormahy dissociating &-am&x o group in the native protein. On the basis of the known” amino acid composition of ribonuckase and the equation of Li@erstromLang and Nielsen 12,the pi value in water (9.633 can be explained by assuming that three out of six phenolic groups are dissociated &K = 9.95);w~e.tfie’mean.pKvalue of the ten &-amino groups, considered to be identical, is 10.22.These t+res agree with those required for the explanation of the entire titration cu~&~. -T. _. . The absence of a signihcant shift in pIunder the action of 6 M urea should then be interpreted by assuming that the protein is barely denatured under-the particular experimental conditions (at 4O and pH 9-l 1) and that an eventuahy wurring part&I denaturation do& not influence the degree of dissociation of the mentioned pi determining protoIytic groups. The results of Nelson and :HummeiZL (dens&ration by
8 M urea at 25” and pM 7.3 is incomplete) and viscosity and opti& rotatory dis-in urea ,vd guauidine hydro&oride,~sofutions are consistent with this interpretation. Moreover, it. is well known~ that polyhydric alcohols (in this instance sucrose) protect the native conform&ion of ribonucfease .against denaturation by urea. In the presence of ethanol, which is known 23*24.to have a de&b&zing effect on the native conformation of ribonuclease, the p1 value differs si.gnikaMy from-.that in water. We ascribe this pl shift to a general increase in the pK values of s-amino groups upon denaturation. This increase, which is consistent with the fact that the persion data for ribonuclea&
-of.
pK values of side-chain amino groups required to explain the titratii~!~~mtrves’ native proteins are generally smaher than expected, was asc+ed by Tzmf~rci~~ to the hydrophobic&y of the major parts of lysine sidechains. these parts thereby&%td to be buried in the interior of the native structure, resuhing in stabifi+ion -of the urtcharged form of the e-amino groups reIative to the char@ form and hence -in a de-
crease in their pKvaIue. This view was confirmed by Nozaki andkanford, who showed that the pK values of amino groups neede&to explain .thktit&ion mes of an&o acids26and proteins1g in 6 Ri _“uanidine hydrochloride are close to the corresponding pK values of amino acids in aqueous dilute salt solution. Consequentiy, we catcmated the mean pK’vahk of the ten .&-amino groups (considered to be identical) needed to expiain the plvalue (9.85) f&cl in 30% ethanol assuming all other pK values to be unchanged relatkto their e&ctive~v&es in the
180
W. J. GELSEMA,
C. L. DE LIGNY,
N. G. VAN DER VEEN
native state. We obtained the result pK = 10.55, a value intermediate between the “expected” value acecording to Tanfordz5 (10.4) and that (10.8) for lysine in aqueous solution25. The p1 value (Table III) of b-lactoglobulin, i.e., the approximately equimolar mixture of the genetic isomers A + B, calculated from measurements in a sucrose gradient, is slightly lower than expected from literature data_ The latter are not consistent, however. By means of isoelectric focusing the following values are found: -5.26 (A)’ and 5.34 (B)‘; 5.21 (A)2’ and 5.34 (By’; 5.13 (A)18 and 5.23 (By; and 5.24 (A)1g and 5.14 (A+By. The isoionic point of the protein (AS-B) in pure water was found at pH 5.19 and to be invariable with ionic strength by Cannan et QZ.~‘,but at pH 5.39 and to decrease with increasing ionic strength by Nozaki et &J2. pl values found by moving boundary electrophoresis are genera& lower: 5.19 (ref. 33) and 5.10 (ref. 34). By means of the equation of Linderstrerm-Lang and Nielsen= and the pK values required for an adequate descriptiorP of the titration curve of #?-lactoglobulin (A+B) in aqueous solution, a pl value of 5.36 can be calculated. The p&, -pl correction terms are large in this instance, so the only comment that can be ‘made on the p1 shifts exerted by urea and ethanol is that they are small. A small effect is indeed expected. From the work of Tanford and co-workers25*3r, it is -known that two out of fifty-three carboxyl groups are abnormally weak (pK & 7.4) in the native protein but behave as “expected” (pK M 4.8) in the denatured state. Calculation shows that this conformation change upon denaturation should result in a decrease in p1 of 0.07 pH unit. CONCLUSIONS
(1) Pea,,-pl values in sucrose-urea-water and glycerol-ethanol-water mixtures depend on the acidity/basicity and the chemical type of the ampholyte and on the solvent composition. (2) PI,,,-pi vaiues in these mixtures account for the primary medium effect and the pH .measuring cell effect on the isoelectric point of Ampholines. (3) PI,,, -pl values for Ampholines can be used to correct the apparent isoelectric points of proteins measured in these media, giving pl values which are useful for conformational studies. REFERENCES 1 2 3 4 5 6 7 8
%“. J. Gelsema and C. L. de Ligny, 1. Chromafogr.. 130 (1977) 41. W. J. Gelsema, C. L. de L&y and N. G. van der Vezn, J. Chrowazrogr.,140 (1977) 149. W. J. Gekema. C. L. de Ligny and N. G. van der Veen, J. Chrumarogr., 154 (1978) 161, C. Tanford, C. E. Buckley, III, P. K. De and E. P. Lively, 1. Biol. C&m., 237 (1962) 1168. C. Tanford, A&an. Profein Chem., 23 (1968) 122. 1% Ui, Biochim. Biophys. Acta, 229 (1971) 567. M. R. Salaman and A. R. Will xamson, Biochem. J., 122 (1971) 93. R. V. Jkphson, S. K. Mah-, C. V. Morr, R. Jennessand R. K. Lindorfer, AI& Biocfrem_, 40 (1971) 476. 9 P. G. Righetti, E. Gianaza, 0. Brenna and E. Galante, J_ Chronrarogr., 137 (1977) 171. 10 F. Franks and D. Eagland, CKC Crir. Rev. Biuchem., 3 (1975) 165. 11 A. V. Lebedev, V. P. Neuciachin, V. G. Zima and V. G. Ryadchikov, Ski. ToaL, 23, No. 2 (1976) 22.
ISOELECFRIC
POKNTS OF PROTEINS
ISI
12 K. Lindast~~m-Lang and S. 0. NieIsen, in M. Bier Editor), ffkfrq&o&s, k&emic l?cess,. New YoFk, 1959, al. 5 p_ 35. 13 G. R Stark, W. H. Stein and S. Moore, J_ B&l. C&m_, 235 (1960) 3177. 14 W. J..Gclsema, C. L. de Ligny. A. G. Ran&se and H. A. Blijkverz, Rec. Trav. Ci’Ztt. Pajs-.&, 85 (1966) a7. 15 W. J. Gekema, C. L. de Ligny and G. F. Viserman, Rec. fiav. C&m. Pays-Bas, 84 (1965) 1129 16 Q_ S. T&in, Rev- Brcs. Pea.@_ A&G?_ Biol., 3 (1970) Sf_ 17 J. W. Donovan, M. Laskomki, Jr., rtnd H. A. Sheraga, 3. Mot: Biai.., 1 (1959) 293:. It? M. Noelken and M. Reibstein, AreIt_ Bidrem. Biuphys., 123 ($968) 397. 19 Y. Nozaki and C. Tanford, 3. Amer. Ciiem Sk., 89 (%%I) 742. . 20 C. Tanford and J. D. Hauenstein, /. Amer. Chem. Sac., 78 (E956) 5287. 21 D. 6. Smyth, W. H. Stein and S. Moore, & &M C&m.., 238 (1963) 227. 22 C. A. Nelson and J. P. Hummel, J. 5rX. Chem., 237 (1962) 1567. 23 S. Y. Gerlsrna, J. Bioi. Chem., 243 (1968) 957. .24 S. Y. GerZsrnzand E. R. Stuur, Int_ I. PepticieProtein Res., 4 (1972) 377. 25 C. Tanford, A&m. Protein Ckem., 17 (1362) 69. 26 Y. Nozaki and C. Tanford, J. Amer. Chem. Sac., 89 (1%7) i36_ 27 L. J. tip!an and J. F. Foster, Bkkhernfsslry.10 (1971) 630. 28 S. Fredrikssoe, Am&_ Bk&em., 50 (1972) 575. 29 N. Catsknpoofas, Anal. Biockem.,,54 (1973) 66. 30 B. J. Radola, Bhchim. Brbphys_Acta, 295 (1973) 412 31 K. Cannan, A. I%. paimer and A. C. Kibrick, J. Bioi. Clrem., 142 (1842) 803. 32 Y. Nozaki, L. G. Bunville and C. Tanford, J. Amer. Ch. Sbc., 81 (i959) 5523. 33 K. 0. Pedenen. Bfodrem. J., 30 (1936) 961. 34 C. H. Li, J. Amer. C/km. Sot., 68 (1946) 2746.
.”
11,550
ISOELECTRIC POINTS OF PRO-@INS, DETER&NED FOCUSING IN THE PRESENCE‘QF UREA AND ETH~OL
.. .
W. J. GELSEMA,
C. L. DE LIGNY
and N. G. VAN
DEEC VE&
Analyiica Ciiern~Oy hboralory. Sfate UnmerSfy Wt~eciit,Croesstm&~~tz, &ree&&(The Nerhcr; [&.I (Fust received May lOth, 19s; revised manuscript re&ved September 29$1918)
.I ‘. . SUMMAFW
..
-_
-_ ‘.
rsoetectric points, plep9, in sufxose-urea-wter and gfycerokhano@i+% mixtures and isoekctric points, PI, in water have been dete~ed at 29 foriscarrier ampholytes. ‘T&edifferences,pf,, -pI, are shown to account fqr the prnqaq mediumeffkctand the pH measuringcelfeEectonthe i&ekctric‘point~Thedifiken~~s, protf&s. _ Pl,,, -pi, for Ampholiks are used to correct apparentisoektric points pl shifts resulting from the denaturing effket of urea and et&&31 are diQ+sed in terms of the conformation change_
of
INTRODUCTiON In a recent series ofpapersf3, we showed that the apparent isoekctri~ #z&s, of proteins, measured by isoelectric focusing in densiw gradients of SUCRIS% .$&ol or ethy!ene glycol in the presenceof mrrier amph&tes, can irec&e&d fk the primary medium effect and the pH mezxsuring-CeEf effect tg giv&i&kAri~ points;pi, in water. The correctionterms to&? used, yhich wereta+~Iate@~,depend 06 p&,&i the concentration of l he non-electrolyte, the tempetatirre&d,the &emicaE nature of the carrierampholyte, fn this approach, it was tacitly assUmedthat t& c~nforni&m ,. of proteins is not affected by the pdient-forming non&x-tro~ytL T&e is; indeed, enough evideke4~’ in favour of this assumption with sacrose, -g&&of-and ethylene gIyco1at the concentrations used in isoekctric fkxising. In a paper dealing with the effect of urea on the behavitiurof so&e pro&ins in ’ isoelectricfocusing, UP demonstratedthe value of measurementsof isoelectricpoin& in the absence and presenceof urea for conformationaEstudiesof proteink-However, : ’ in that paper, isoekctric points measured in the presenceof u&k we& ?Q& foi the primarymedium effect and the pH measuringcell effect due to t&d?.& aqekge correction term, independent of p&,1 In- view of the .evidenceres&i~~g.f?om ow wodP, this procedure appears to be iiicorrectl. Moreover, literates W&ES ofax-. rection terms, pl,,, -pI, associated with the effkd of WC& k&e a. cotid+a$Ie : i scatter,as is shown in Table I. As we agree with W on the V&I& of isue!ectk&using for conformatikkaf
W. J.
172
TABLE I LITEE+4TURE VALUES OF PI.,, Urea concrntration (Ml
Correction PL -PI
6 6 7 7
0.42 0.3; 0.4 0.9 0.35
-
GELSEMA, C. L. DE LIGNY, N. G. VAN DER VEEN
pZ
term,
USED TO CORRECX FOR THE EFFECT OF UREA Range of PI.,, for which correction term is stated to be valid
Reference
4-10
4-6; 7-10 3-6 2.5-6
8 9
studies of proteins, we determined the appropriate correction terms accurately. For p1 for Ampholines in the presence of two this purpose we measured values of PI,,,reagents that have a conformation-changing effect on proteins, viz., urea and ethanol*, as a function of the concentration of these reagents and of the PI=,,, values of the Ampholines. As the effect of urea or ethanol on the conformation of proteins can be studied by density gradient isoelectric focusing, these PI,,,-plvalues were determined also as a function of the concentration of two gradient-forming non-electrolytes, viz., sucrose and glycerol. Supposing that urea and ethanol do not affect the conformation of Ampholines, these values of PI,,, -pl can be assumed to account correctly for the primary medium effect and the pH measuring cell effect of the reagents on the isoelectric point of a pr&ein. Any difference between the isoelectric point of a protein in water and that obtained by correction of the pl,,, value measured in the presence of the conformation-cha’nging reagent can then be associated with the conformation change of the protein. It should be borne in mind, however, as was pointed out by UP, that the absence of a pl shift due to a conformation-changing reagent cannot always be interpreted in terms of retention of the native configuration, as the pl value is informative only for the degree of dissociation of a few protolytic groups. As a test of the validity of the correction procedure, we reappraised the literature values of isoelectric points of some proteins in the absence and presence of urea and measured the isoelectric points of two proteins, viz., ribonuclease and p-lactoglobulin, in the absence and presence of urea and ethanol. In those instances where a significant shift in corrected isoelectric point was found, this shift was interpreted in terms of the conformation change by comparing the result with calculations on the basis of the equation of Linderstmm-Lang and Nielsen’“, using the known chemical compositions of the proteins in question.
While urea and ethanol are both frequently used in conformational studies of proteiz”, cnly the former has been used as such in conjunction with isoelectric focusing. There is. however, no major objection to the use of ethanol in isoelectric focusing for conformaticnal stcdies. Practical drawbacks are, of course, its low density, which induces a lower stability of glycerol density gradients. and its high volatility, which can be expected to render isoelectric focusing experiments cn granulated gels more dificult. To our knowledge, such gel-isoelectric focusing experiments hale net teen carried out; experiments in a glycerol gradient, containing up to 60% of ethanol, have heen Ferfcrmed by Letedev et al.” for the separation of water-insoluble ccm proteins. l
i.WELECTRX
PQXNTS OF PROTEINS
173
EXPERIMENTAL
Measurements of isoelectric points of ‘Carrier ampholytes
The isoelectric points of 2 % (w/v) solutions of Amphoiines (LKB, Stockholm, Sweden) and Servaiyte (Serva, Heidelberg, G.F.R.) in water (pr) and in the presence of urea, ethanol, urea + sucrose and ethanol f glycerol (p1,,,) were determined at 25” as described eariieti. The chemicals used were urea (Merck, Darmstadt, G.F.R., Cat. No. 8488), ethanol (Baker, Phillipsburg, N-J., U.S.A., anaiysed grade), sucrose (Baker, analysed grade) and glycerol (Merck, p-a. grade). The specific conductivity of an aqueous 6 M solution of urea was 9.7 - 10-6R-s.cm-1 at 25”, indicating the absence of eiectrolyites. Urea was dissolved just before the measurements without raising the temperature above 25”, to prevent the formation of cyanate’“. At relatively high concentrations of ethanol the reduced soiubiiity of the carrier amphoiytes eesulted in turbid solutions. Measurements of isoelectric points of proteins
Aliquots of 15 mg of bovine pancreatic ribonuclease (Sigma, St. Louis, MO., U.S.A., Cat. No. R-4875) OF bovine B-iactogiobuiin (Serva, Cat. No. 27440) were focused in au elect&focusing coiuruu (LKB 8100-l) ‘at a temperature of the cooling water of 4”. The concentration of Amphoiines (pH range 9-l 1 for ribonuclease aud 3.5-10 for @-iactogiobuiin) was 2% (w/v). Density gradients were produced with a gradient mixer (LKB 8121) and ranged from 50 to 5 % (w/w) of sucrose in the absence and from 42 to 4% (w/w) of sucrose in the presence of 6 M urea, and from 60 to 6 % (w/w) of glycerol in the absence and presence of 30 % (w/w) of ethanol. The cathoiyte was a 0.25 M sodium hydroxide solution; the anolyte was 0.16 M orthophosphoric acid OF 0.01 M acetic acid. After focusing at constant power (5 W) for 48 h (LKB 2103 power supply), the contents of the column were collected in fractions of 3 ml for ribonuclease and 1.5 ml for ,8-iactogiobuiin. The extinction at 280 nm of these fractions was measured using a Vitatron Type MPS spectrophotometer. Z&z pH at 25” of the fractions with maximum UV extinction was measured, and the sucrose OF glycerol content was determined from the refractive index. We used calibration graphs established with solutions of 2% (w/v) Ampholines and 6 &1 urea in sucrose-water mixtures of varying sucrose content and with solutions of 2 oA(w/v) Ampholines and 30 oA(w/w) ethanol in glycerol-water mixtures with varying glycerol contents, respectively. RESULTS
Isoelectric points of carrier ampldytes The results are given in Table II. As an example, Figs. 1 and 2 give plots of PI,,* -pl versus PI,,, at a constant concentration (6 M) of urea in sucrkc-water
mixtures and at a constant concentration (30 %, w/w) of ethanol in glycerol-water mixtures, respectively. Figs_ 3 and 4 show values of 1p,,,-p1 ~ers~.spf,,, in aqueous solutions of urea and ethanol, respectively. Isoelectric points of proteins
The results for pl,,,
at 25” are given in Table III. Table III also includes pl
RANGESANDFORSERVALYTEOFpHRANGE2-4
(%,1d11))
ngertt
.I
-
Glycerol(O%)(1.33) (20%)(1.75)
Ethanol (72%,w/w)
1
Glyccrol(O%)(0.75) (20%)(1.00) (40%)(1.38)
Ethanol (5O%,w/w)
_..
Glycerol(O%)0.38 (20%) 0.51 (40%) 0.71 (60%) 1.04
Ethanol (30%,w/w)
-.
Sucrose (0%) 0.85 (15%) 0.89
Urea(9M)
0.56 0.58 0.62 0.68
Sucrose(O%) (15%) (30%) (45%)
Urea(6M)
. ._. ._
(1.38) (1.75)
073 0,95 1.34
0.36 0.50 0.70 1.06
0.74 0.80
0.47 0.49 0.54 0.62
-_
-
0.62 0.81 I.13
0828 0,40 0.59 0.90
0.66 0.71
_ -
. -
(0.76) 0.87 __.._.. __.
0.33 0.45 0.65
0,09 0,17 0,29 0.47
0.52 OS5
0.33 0,34 0,37 0.42
0.16 0.15 0.16 3.19 0,23
0.20 O,l9 0.21 0.24 0.29 0.41 0.44 0.48 0.55
pfl(S-7) pl5.94
.~1
.__.,.._
.
.
(0.61) 0.60
0.30 0.38 0.48
0.11 0.17 0.25 0.34
OS2 0.56
0.33 0,35 038 0.45
0.16 0016 0,17 0.21 0.26
-
pli (6-8) ~16.90
.__._. _.___. .-._
pli(4-6) p14.87
,_.-.... -- --. .._..__ -_.._.._.. ___. .__..,__.
Antplfolitw
pH(2.5-4) pH(3,5-5) pI4.34 ~12.90 .---- --__-. ..._._ ..- .__._, ____.-.__.._..___-- .._- .-._. ._-._ Urea(3M) Sucrosc(O%) 0.27 0.21 0.21 (15%) 0.27 0.23 (30%) 0.27 0.26 (45%) 0.30 0.32 (55%) 0.33
cfmerrrrrttbtt
-.-.
-
-.
...-...
----
0.57 0.61
0.36 0.38 0.40 0.47
0.17 0,17 0,17 0.20 0.24
_.__
_._..
0.26 0.33 0.43
0.08 0.13 0.21 0.31
0.58 0.60
0.35 0.37 0.38 0.41
0.17 0.16 0.15 0.15 0.17
..---
-0.02 0 0.06
-0.08 -0.07 -0.05 0.01
0.50 0.49
0,31 0.30 0.26 0.24
0.15 0.11 0.07 0.02 0.01
--
.._
(1.13) (1.46)
(0.60) (0.83) (1.20)
0.26 0,38 0.56 0.89
0.80 0,84
0,53 0.55 0.58 O-66
;H(2-4) pl3.40 .._-.... ., 0.27 0.26 0026 0.2g 0,33
lyre
Servn-
__...,_
-_.
..
.
_.. .._ .. ..-........
.
p11(7-9) pN(8-9.5) pH(9-11) ~18.03 ~18.85 ~19.76
.I
was nradcina turbid solution. inpnrcnlhcscs dcnotcthatthecorresponding measurement of PI,,,,,, r&l - pf values ..-. ..-. ..---. .._.. _._.,__ _. .._ ._....__.._ ___: .._._ . ..__-- ,.. Ditratrrritrg GrflflierrrCirrier ~trrpholyte ._. ._. _. ..-._-_ . . ..- ._-.. ..-.. . -. ._ ._- ._. ngerit arrfl jiwtfrlll~
VALUES OF PI,,,, - pfAT25"FORAMPHOLINL?SOFVARlOUSpH
TABLE11
175
ISOELECTRIC POW-i-!3 OF PROTEINS
P&.)-PI
11: wo-
Ox)-
Fig. 1. Values of PI.,, - pl verse pl,,, for Ampholines (open symbols) and Servalyte (closed symbols) at 25” in 6 &l solutions of urea in water (a) and sucrose-water mixtures containing 15 (a), 30 (8) and 45% (w/w) (n) sucrose.
values at 25” calculated by applying correction terms, pz,, -p1, resulting from previous work3 and Table II in this paper. DISCUSSION
The results in Table II and Fig. 3 corroborate our prediction that the effect of urea on the apparent isoelectric point of an ampholyte depends on the acidity of the ampholyte. The same conclusion holds for ethanol (Fig. 4). Also in accordance with
120
.
too 0.80 / -
I
0.60 -
Phppjl
0.40 -
0200 _________
4201 3.
_ ____
4
5
_ _____
7
6
-_
_ __________
8
9
m-e_
10
PIwJ
Fig. 2. Values of PI.,, - pl versusPI.,, for Ampholines (open symbols) and Servalyte (closed symbols) at 25” in 30% (w/w) solutions of ethanol in water (0) and glycerol-water mixtures containing 20 (a), 40 W) and 60% (w/w) (A) plyceroi.
176
W. J. GELSEMA,
01
3-
4
5
6
C. L. DE LIGNY,
6
7
9
N. G. VAN DER VEEN
10
11
PbP
Fig. 3. Values of PI.,, - pl versus PI,,, for Amphohnes (open symbols) symbols) at 25” in aqueous solutions containing 3 (a), 6 (0) and 9 M (A)
and ServaIyte (closed urea.
PI,pp,-PI
-a20
j
4
5
6
7
8
9
10
%PD
Fig. 4. Values of PI,,, - pl versus pl.,, for Ampholines (open symbols) and Servalyte (closed symbols) at 25” in aqueous solutions containing 30 (S), 50 (o) and 72% (w/w) (A) ethanol.
TABLE
III
ISOELECXRIC
POINTS
OF PROTEINS
(25”)
Protein
Conformationchanging agent
&P
Gradient-forming agent (36, w/w)
PI,,,
Ribonuckase
-
9.50 9.53 9.Qo 9.75 9.81 9.82 4.98 4.99 5.40 5.38 5.76 5.74
38.6 % sucrose 40.7 % sucrose 27.5 y0 sucrose 46.5 % glycerol 48.6 % glycerol 47.8 % glycerol 34.2 y0 sucrose 34.8 % sucrose 28.0 % sucrose 28.2 0/0sucrose 49.3 % glycerol 47.3 % glycerol
-0.11 -0.12 0.28 -0.04 -0.05 -0.06 -0.04 -0.04 0.47 0.47 0.68 OX%
&Lactoglobulin
6Murea 30% (w/w) ethanol 30% (w/w) ethanol 30% (w/w) ethanol 6Murea 6Murea 30 % (w/w) ethanol 30 “/6 (w/w) ethanol
- pI
pl 9.61 9.65 9.62 9.79 9.87 9.88 5.02 5.03 4.93 4.91 5.08 5.10
ISOELECTRiC
POTN-iS OF PROTEINS
177
earlier findings3, the effects of urea f sucrose and ethanol + glycerol (see Figs. I and 2) on the pl,,, of Servalyte differ from those on the PI,,, of Ampholines of comparable acidity. A comparison of values of pZ*,, -pZ in sucrose-water and glycerol-water mixtures3with the results in Table II shows that the effects of sucrose and urea and of glycerol and ethanol, respectively, are not additive. As there are no literature data on the pK values of weak acids in ternary solvent systems, this finding cannot be commented upon. The general appearance of the curves in Figs. 1 and 2, which show the effect of sucrose and glycerol, respectively, is similar to that in Figs. 3 and 4, which show the effect of urea and ethanol, respectively, and to that in Fig. 1 in ref. 3, demonstrating the effect of ethylene glycol. This suggests that the pl,,, -pZdifferences due to the five mentioned non-electrolytes are caused by the same effects, viz., the primary medium effect and the pII measuring cell effect. As was pointed out in a preceding pape?, by combining values of pZ,,,-pZ with values of 6, accounting for the pH measuring cell effect, values of pZ’-plcan be calculated : pr-pZ
= pZ,,,-pZ--6
Subsequently it was shown in that pape$ that the pZ’-pZ values of Ampholines calculated in this way arc in between the pK’-pK values of carboxylic acids and alkylsubstituted ammonium ions, giving additional support to the view that the pZ,,,-pd differences of Ampholines due to sucrose, glycerol and ethylene glycol are indeed caused by the two effects mentioned above. 6 values due to ethanol are known lo_Therefore, pZ*-pZ values were calculated -0 5 with literature valuesis of pK’-pK. An analogous comand are compared in Ft,. parison for the effect of urea cannot be given, owing to the lack of corresponding 6 values. One can compare, however, (see -Fig. 6) the pl,,,-pZ values of Ampholines with literature values of pK,,, -pK for acetic acidI and the n-butylammonium ion.“. Figs. 5 and 6, which should be compared* with Fig. 3 in ref. 3, show that the effects of both urea and ethanol on the isoelectric points of Ampholines are indeed intermediate between the effects of these non-electrolytes on the dissociation constants of carboxylic acids and alkyl-substituted ammonium ions. We conclude, therefore, that the pZ,,,- pZ values in Table II can be used to correct for the primary medium effect and the pH measuring cell effect on the isoelectric points of proteins. Therefore, these values were used in a reappraisal of the literature values of the isoelectric points of some proteins, measured by isoelectric focusing in the presence of urea (see Table IV). The data, confined to proteins that are not dissociated into subunits by the action of urea, clearly indicate the importance of the correction procedure. For insulin and haemoglobin, there is no siguificant influence of urea on the pZ value, which leads to the conclusion that these proteins contain no abnormally dissociating groups, as far as groups having an influence on the isoelectric point are concerned. For a comment on this conclusion, which was also arrived at by Ui”, we -__ * For that reason,data on propionicacid and n-propyiamine are includedin Fig. 5.
W. J. GELSEMA,
178
C. L. DE LIGNY,
0
I
2
N. G. VAN DER VEEN
4
6 _a x; Molarityof lreo
Fig. 5. Values at 25” of pK* - pK for acetic acid (e), propionic acid (A), n-butylamm&ium ion (I) and n-propylammonium ion (V) and PI* - pl for Amph~lim~, pi = 2.90 (0) and p1= 9.76 (a), in ethanol-water mktures.
Fig. 6. Values at 25” of pk.,,
- pK for acetic acid (e) and n-butylammonium ion (a) and p&, and pZ = 9.76 (o), in aqueous urea solutions.
-
pZ for Ampholines, pZ = 2.90 (0)
TABLE
IV
LITERATURE OF UREA
VALUES
Protein
Urea concenfration
--.-
__--._____
OF ISOELECTRIC PL,,
WO)
l-M)
l
POINTS IN THE PRESENCE Concentration of sucrose f s, w/w) --
PI,,,
-
pl
AND ABSENCE
pI(2Y)
Reference
Insulin
0 6
5.69 6.15
20 20
-0.01 0.36
5.70 5.79
6 6
Haemogfobin
0 6
7.07 7.50
38 34
0.01 0.40
7.06 7.10
6 6
Ribonuclease
0 6
9.26 9.93
45 45
-0.08 0.26
9.34 9.67
6 6
a-Czsein
0 7
4.4”’ 4.66
-20
-0.8
4.4 3.9
8
0 7
4.5”’ 5.4
-20
-0.8
4.5 4.6
8
0
3.7; 4.1 ** 6.2
-20
-0.8
3.7; 4.1 5.4
8
/Kkein li-Casein
7
l
-
* PZ,~, (25”) was obtained from the literature value p&t) using~pl/ilTvalues equal to the mean of the distribution of dpK/dT values given in Fig. 2 in ref. 3. ** Estimated from the descriptions of the focusing experiments and the graphs of the focusing patterns. *** Moving boundary values.
ESOEFBXRIC
POINTS
OF
PROiEMS
-179
refer to his paper. For ribonuckas&, however, the significant shift in &due to .urea would imply the presence of abnormally dissociating groups in the native .stat$. The data on caseins show, contrary to the conclusion th& would be obtained Corn a comparison of p&,, values, that a-casein does and /kasein does not containanomalously dissociating groups determining the isoektric point in the native state_ This. is consistent with the evidence from viscosity measurements~*,indicating that ‘&case&r is a random coil in the native statti. :.:: In Table III, our experimental resdts on dboauckase are given. Whereas the value for pIestimated from the measurements ~FI 6 M urea agrees with thaffound.by UP, the p1 value found in the absence of urea differs _mnSiderabIy from his vafue (9.34). Our rest&s are in good agreement with the.pH of aiqueoussoiutions of rib& nuclease, found by Nozaki and ‘+SordLg (9-60 at zero.ionic strength and 9.71 in 0-U M potassium chloride solution) and by Tanford and IIauensteirP (9.65 in O;LS M. potassium chloride sohttion). We conclude therefore, in contrast to -UP,. that .6 A& urea does not produce a signScant pi shift_ Further, there is no need for he assump tion ofan abnormahy dissociating &-am&x o group in the native protein. On the basis of the known” amino acid composition of ribonuckase and the equation of Li@erstromLang and Nielsen 12,the pi value in water (9.633 can be explained by assuming that three out of six phenolic groups are dissociated &K = 9.95);w~e.tfie’mean.pKvalue of the ten &-amino groups, considered to be identical, is 10.22.These t+res agree with those required for the explanation of the entire titration cu~&~. -T. _. . The absence of a signihcant shift in pIunder the action of 6 M urea should then be interpreted by assuming that the protein is barely denatured under-the particular experimental conditions (at 4O and pH 9-l 1) and that an eventuahy wurring part&I denaturation do& not influence the degree of dissociation of the mentioned pi determining protoIytic groups. The results of Nelson and :HummeiZL (dens&ration by
8 M urea at 25” and pM 7.3 is incomplete) and viscosity and opti& rotatory dis-in urea ,vd guauidine hydro&oride,~sofutions are consistent with this interpretation. Moreover, it. is well known~ that polyhydric alcohols (in this instance sucrose) protect the native conform&ion of ribonucfease .against denaturation by urea. In the presence of ethanol, which is known 23*24.to have a de&b&zing effect on the native conformation of ribonuclease, the p1 value differs si.gnikaMy from-.that in water. We ascribe this pl shift to a general increase in the pK values of s-amino groups upon denaturation. This increase, which is consistent with the fact that the persion data for ribonuclea&
-of.
pK values of side-chain amino groups required to explain the titratii~!~~mtrves’ native proteins are generally smaher than expected, was asc+ed by Tzmf~rci~~ to the hydrophobic&y of the major parts of lysine sidechains. these parts thereby&%td to be buried in the interior of the native structure, resuhing in stabifi+ion -of the urtcharged form of the e-amino groups reIative to the char@ form and hence -in a de-
crease in their pKvaIue. This view was confirmed by Nozaki andkanford, who showed that the pK values of amino groups neede&to explain .thktit&ion mes of an&o acids26and proteins1g in 6 Ri _“uanidine hydrochloride are close to the corresponding pK values of amino acids in aqueous dilute salt solution. Consequentiy, we catcmated the mean pK’vahk of the ten .&-amino groups (considered to be identical) needed to expiain the plvalue (9.85) f&cl in 30% ethanol assuming all other pK values to be unchanged relatkto their e&ctive~v&es in the
180
W. J. GELSEMA,
C. L. DE LIGNY,
N. G. VAN DER VEEN
native state. We obtained the result pK = 10.55, a value intermediate between the “expected” value acecording to Tanfordz5 (10.4) and that (10.8) for lysine in aqueous solution25. The p1 value (Table III) of b-lactoglobulin, i.e., the approximately equimolar mixture of the genetic isomers A + B, calculated from measurements in a sucrose gradient, is slightly lower than expected from literature data_ The latter are not consistent, however. By means of isoelectric focusing the following values are found: -5.26 (A)’ and 5.34 (B)‘; 5.21 (A)2’ and 5.34 (By’; 5.13 (A)18 and 5.23 (By; and 5.24 (A)1g and 5.14 (A+By. The isoionic point of the protein (AS-B) in pure water was found at pH 5.19 and to be invariable with ionic strength by Cannan et QZ.~‘,but at pH 5.39 and to decrease with increasing ionic strength by Nozaki et &J2. pl values found by moving boundary electrophoresis are genera& lower: 5.19 (ref. 33) and 5.10 (ref. 34). By means of the equation of Linderstrerm-Lang and Nielsen= and the pK values required for an adequate descriptiorP of the titration curve of #?-lactoglobulin (A+B) in aqueous solution, a pl value of 5.36 can be calculated. The p&, -pl correction terms are large in this instance, so the only comment that can be ‘made on the p1 shifts exerted by urea and ethanol is that they are small. A small effect is indeed expected. From the work of Tanford and co-workers25*3r, it is -known that two out of fifty-three carboxyl groups are abnormally weak (pK & 7.4) in the native protein but behave as “expected” (pK M 4.8) in the denatured state. Calculation shows that this conformation change upon denaturation should result in a decrease in p1 of 0.07 pH unit. CONCLUSIONS
(1) Pea,,-pl values in sucrose-urea-water and glycerol-ethanol-water mixtures depend on the acidity/basicity and the chemical type of the ampholyte and on the solvent composition. (2) PI,,,-pi vaiues in these mixtures account for the primary medium effect and the pH .measuring cell effect on the isoelectric point of Ampholines. (3) PI,,, -pl values for Ampholines can be used to correct the apparent isoelectric points of proteins measured in these media, giving pl values which are useful for conformational studies. REFERENCES 1 2 3 4 5 6 7 8
%“. J. Gelsema and C. L. de Ligny, 1. Chromafogr.. 130 (1977) 41. W. J. Gelsema, C. L. de L&y and N. G. van der Vezn, J. Chrowazrogr.,140 (1977) 149. W. J. Gekema. C. L. de Ligny and N. G. van der Veen, J. Chrumarogr., 154 (1978) 161, C. Tanford, C. E. Buckley, III, P. K. De and E. P. Lively, 1. Biol. C&m., 237 (1962) 1168. C. Tanford, A&an. Profein Chem., 23 (1968) 122. 1% Ui, Biochim. Biophys. Acta, 229 (1971) 567. M. R. Salaman and A. R. Will xamson, Biochem. J., 122 (1971) 93. R. V. Jkphson, S. K. Mah-, C. V. Morr, R. Jennessand R. K. Lindorfer, AI& Biocfrem_, 40 (1971) 476. 9 P. G. Righetti, E. Gianaza, 0. Brenna and E. Galante, J_ Chronrarogr., 137 (1977) 171. 10 F. Franks and D. Eagland, CKC Crir. Rev. Biuchem., 3 (1975) 165. 11 A. V. Lebedev, V. P. Neuciachin, V. G. Zima and V. G. Ryadchikov, Ski. ToaL, 23, No. 2 (1976) 22.
ISOELECFRIC
POKNTS OF PROTEINS
ISI
12 K. Lindast~~m-Lang and S. 0. NieIsen, in M. Bier Editor), ffkfrq&o&s, k&emic l?cess,. New YoFk, 1959, al. 5 p_ 35. 13 G. R Stark, W. H. Stein and S. Moore, J_ B&l. C&m_, 235 (1960) 3177. 14 W. J..Gclsema, C. L. de Ligny. A. G. Ran&se and H. A. Blijkverz, Rec. Trav. Ci’Ztt. Pajs-.&, 85 (1966) a7. 15 W. J. Gekema, C. L. de Ligny and G. F. Viserman, Rec. fiav. C&m. Pays-Bas, 84 (1965) 1129 16 Q_ S. T&in, Rev- Brcs. Pea.@_ A&G?_ Biol., 3 (1970) Sf_ 17 J. W. Donovan, M. Laskomki, Jr., rtnd H. A. Sheraga, 3. Mot: Biai.., 1 (1959) 293:. It? M. Noelken and M. Reibstein, AreIt_ Bidrem. Biuphys., 123 ($968) 397. 19 Y. Nozaki and C. Tanford, 3. Amer. Ciiem Sk., 89 (%%I) 742. . 20 C. Tanford and J. D. Hauenstein, /. Amer. Chem. Sac., 78 (E956) 5287. 21 D. 6. Smyth, W. H. Stein and S. Moore, & &M C&m.., 238 (1963) 227. 22 C. A. Nelson and J. P. Hummel, J. 5rX. Chem., 237 (1962) 1567. 23 S. Y. Gerlsrna, J. Bioi. Chem., 243 (1968) 957. .24 S. Y. GerZsrnzand E. R. Stuur, Int_ I. PepticieProtein Res., 4 (1972) 377. 25 C. Tanford, A&m. Protein Ckem., 17 (1362) 69. 26 Y. Nozaki and C. Tanford, J. Amer. Chem. Sac., 89 (1%7) i36_ 27 L. J. tip!an and J. F. Foster, Bkkhernfsslry.10 (1971) 630. 28 S. Fredrikssoe, Am&_ Bk&em., 50 (1972) 575. 29 N. Catsknpoofas, Anal. Biockem.,,54 (1973) 66. 30 B. J. Radola, Bhchim. Brbphys_Acta, 295 (1973) 412 31 K. Cannan, A. I%. paimer and A. C. Kibrick, J. Bioi. Clrem., 142 (1842) 803. 32 Y. Nozaki, L. G. Bunville and C. Tanford, J. Amer. Ch. Sbc., 81 (i959) 5523. 33 K. 0. Pedenen. Bfodrem. J., 30 (1936) 961. 34 C. H. Li, J. Amer. C/km. Sot., 68 (1946) 2746.