O VAPORS BY LYOPHILIZED β-LACTOGLOBULIN ... - ACS Publications

Sorption of FLO and. D20 Vapors by' Lyophilized q-Lactoglobulin. 811 concentration range where the excess solvent would favor solvent rich complexessi...
0 downloads 0 Views 601KB Size
.June, 19GO

SORPTIOS O F H,O

AND

D,O

YAPOKS BY

LYOPHILIZED a-LACTOGLOBULIN

81 1

dynamic activity concepts. Calculation of the activity coefficients for HgBrl in the conveiitional maimer show that at the upper limit of the coiicentrstioii range investigated (m2= 0 10) the activ11Br + HgBr: = [lIHgBrr] = llc + HgBrr- (iv) ity coefficients for HgBrL are 1.022, 1012, 1.008, per mole of solute present. The possibility that 1 . 0 0 ~and 0.999, in the mixtures contaiiiing LiBr, species L121-IgBr4,lIHgBr4-, Ill+, Br- and HgBrr-, XaBr, KBr, RbBr and CsBr, rtspertively The are present in simultaneous equilibria near in- solvent exhibits only small del iatioiis from the finite dilution with a net cryoscopic activity averag- thermodynamically ideal cryoscopir behavior Ahexplanation for the higher v value5 for RhHr iiig to v = 1 as ohserved is not ruled out. The pronounced tendency to form molecular complexes and CsBr is seen in the possibility of wlitl iolutioii may be attributed to the low shielding efficiency“ formation occurring in the mixtures, assuming (Hg+!, 0.67 r f . I)

after reversed exchange treatment of protein:

H-+D-.H.

l-

'4:

1

,

,

2

iP-LACTOGLOBULIN 4 6 Dressure rnm Hg

8

1

1 O a i

Fig. 2. tained was then carefully lyophilized. Five hundred and sixty-four mg. of this protein was weighed into a light glass bucket which was then suspended from a hook a t the lower end of the spring balance. Equilibrium vapor pressures, in the sorption system, were measured by a mercury manometer, using a traveling microscope accurate to zk0.005 mm. All pressure: were corrected by converting the densities of mercury to 0 . The sections of the sorption system containing the bucket and the upper part containing the quartz spring were each water-jacketed. The water, circulrtting through the upper

lized p-lactoglobulin, as plotted against equilibrium vapor pressures a t 17 and 27". The first isotherm, Fig. 1, for H20 was determined at 27". and the adsorption points were obtained up to 3, vapor pressure of 17.86 mm. At) this point the protein sorbed 8.15 mmoles of H20 per gram (14.67% by weight of the dry protein). complete desorption a t 27 the isotherm a t 17" was run. At the completion of each desorption, the weight of the dry protein mas found to be identical with the initial weight a t the start of each run. The early, non-linear, rapid rise in the isotherms changes after the protein has adsorbed 2 millimoles per gram, and becomes essentially a straight line over a considerable range of mpor pressure. An interesting observation may be made a t this point. If the straight lines for both i.otherms are extrapolated back to the zero vapor pressure ordinate, they meet on this ordinate. This suggests a definite relationship to total area coverage at the two temperatures. If the straight-line sections of both isotherms are extrapolated forward, until they cross the ordinates for saturated vapor pressures of H20 a t each temperature (14.53 mm. at 17" and 26.74 mni. a t 27")) then thc intersections show identically the same amounts adsorbed for each temperature. -4s will be proved by later experiments, these hypothetical amounts, adsorbed a t the two saturated vapor pressures, just equal in moles the amount of D20 requirtid to account for the D for H exchange which the experiment? prove takes place when D20 is sorbed by the protein. This seems to be very strong evidence that mono-layer coverage of all of the adsorption sites of the protein for water has not been exceeded until the amounts adsorbed are greater than the values given by the forward extrapolations of the straight-line portions of the isotherms. It may be suggested that the first rapid rise in the isotherms represents adsorption on the most easily accessible sites of the solid protein. The rates of attainment of adsorption equilibrium during the first part of the isotherms are about double that found for the straight lines as well ac: for the following portions of the curves. Folloving adsorption on the more easily accessible sites, the amounts adsorbed 111crease regularly with vapor pressure. It should be noted that the rate of desorption was most (4)

J. G. Fosa and L. H. Reyerson. %bid..61, 1214 (1958).

June, 1960

S O R P T I O S OF

H?O -451) 'DzO YAPORS

BY

LYOPHILIZED (Y-LACTOGLOBULIS

813

rapid a t tlie beginning of this process, becoming very slow as the last vapor was removed. The data for one complete isotherm usually could he obtained in two weeks, an average of 8 hours being required for each equilibrium point. The white, powdery protein showed no observable swelling during adsorption but a slight definite contraction was observed on desorption. These slight volume changes had no effect on the sorption surface area since severd points were rechecked on the straightline sectiov of the isotherms and the amounts sorbed checked the earlier values, indicating complete reversibility in the ranges checked. Gsing the same sample, upon which the H20 isotherms had been completed, 13,O vapor was adsorbed up to a vapor pressure of 10.86 mm. a t 17". It \vas inunediately apparent that the protein adsorbed inore DzO than H20 a t the same vapor pressure. Curve 2 in Fig. 2 gives the data for the adsorption branrh of the first D20 isotherms. Curve 1 diows the H,O isotherm under the same ronditioiis. The D20 isotherm lies above that I-at 27°C.2-at 17°C for HzO throughout its whole course. indicating -adsorption,-- desorption that more D 2 0 molecules were adsorbed by the SORPTION ISOTHERMS OF D 2 0 - v ~ p ow.i ~ protein under like conditions. For example, the /?-LACTOGLOBULIN protein adqorhed more than 12 mmoles of D20 at a vapor pressure of less than 11 mm. compared to , i d :in adsorption of 10 mmoles of H20. 2 4 6 8 IO 12 I4 I6 I9 Pressure rnm Hg. Upon the complete desorption of D20 in the Fig. 3. 'ame manner as for H 2 0 , it was found that the protein had gaiiied 0.832yGof its original weight. brarich of curve 1 of Fig. 1, three additional Curve 3 i i i Fig. 2 is the adsorption branch of the adsorption-desorptions of H 2 0mere carried out with second cornpletP isotherm for D20, using the in- no change in the amounts adsorbed nor in the final creased ntyght of the protein as the zero-weight weights a t the end of each desorption. The fact point at the beginning of the second run. Ppon that the points lie slightly above the original cwmpletioi of tlie srcorid adsorption-drsorptioii adsorption curve suggests the possibility that the isotherm, I hr protein showed a second but smaller deuterium-hydrogen and hydrogen-deuterium exincrease ill weight. Tht. total weight gained at changes may have slightly increased the capacity this time amounted to 1 .OOiypof the original weight. of the protein to adsorb HLO. This might nieati .I third cvniplete iwthtiriii qhowed a further slight srnall structural changes in the dry pmteiii, rcgaiii 111 \\.eight l ' h ~ total me:isured ~ : L I I I i n sultiizg in the niuking of additioiial wrption sites weight antounted to I .0!14?&. Further wrptioni available to H?O. p r o i w ~t11:it thi- gain iii w i g h t duc to the dfviterium These result4 cslearly show that ~-lac~toglohiilnt rwhange ~ v a htlw masinium to bc fornid. C'omqorlx more 1120than HzO at a given ~ a p o rprcbplete isotherms for the wrptioll of 1>?00 1 1 thc sure. The sorbed D20, prior to or at the tinic of tleuterat8td protein were then run at 17 :tnd 27". its desorption. exchanges D for labile hydrogens 'rhe r e d - , :y)pear i l l Fig. 3. 'fhc :dsorptioii in the solid protein. Desorption isotherms show branch oi isoth~rni2 in Fig. :Q was so nrarly identieresiF loops but the adsorbed vapor.: (,a1with :iof Fig. 2 that one further ( ~ i ( ~ l i ithat de~ the maxniiuni deutmuni exchange had heen roached are reversibly desorbed except that a gain iii at the eittl of the third complete sorption of D,O. weight has owurred due to the exchange. dinc~) the maximum exrhange increased thf. weight of Followiiig thc filial tlrhorption of I&()at ? i o ,the protein by an amount equal t o l.09470 of thc i d h e r m 1 of Fig. :5. an :idsorption of HzO waq original w i g h t , the total number of deuterium carried out at relatively high vapor pressure aiid atoms exchanged for hydrogen are (wily deterthis was follon.ed by a complete desorption. At this mined. Since the exchange of one mole of deuterium point the protein showed a definite loss in weight, for hydrogen increases the molecular weight of the indicating that hydrogen was now replacing deuter- protein by 1.0066 g., it is necewary to use an acium in the protein. However, six successive cepted molecular weight for p-lactoglobulin. In adsorption-desorptions of H20 were required t o a recent publication, E. TValdschmidt-Leitzi complete the exchange of hydrogen for the deu- gives the molecular weight of b-lactoglobulin as terium. The protein then weighed exactly the 35,400, while Linderstr@m-Lang6 uses 37,300 in same as it did a t the initiation of the very first his discussion of deuterium exchange on the adsorption of I&O. Water vapor was again ad( 5 ) E. Waldschmidt-Leits, "Chernie der Eineisskoeri~er,"1-erlaq on sorbed b17 this protein at several \-apor pressures. I erdinand En6P Stiitteart. I . 1857. ( ' u r w b iii Fig. 1 shows the first of these results. ( 6 ) K. I.indristroin:-Lang, Souvenir, J. SOC.Bioi. Chenizsfs, India Since the iwtherm lies .lightly above the adsorption 101 (185.5).

: ~

\

, I ;C l I B E R .LSU 1 ) I S T R I B a l l O X Or’ EXCHASGEABLE

TABLE I (LABILE)H Y D R O ~ ~ E I NS THE G fi-L.ACTO(:LoBCLIS

LIOLECL-LE

-

S u n i b e r and kind of groups containing labile hydrogen

51 !I

Pobbible participating unitb in t h e protein

lIole*

-1-H?

-1 H

-SH

-SI

-OH

-8-4(peptide)

COO11

I

.1. Total in the protein backhone (haviirig 3 chains): 300 ( = 320 - l i (proline)) B. Side chains: End groups of the chains Tyrosine Tryptophan Seiine ThreoIiinc. Cysteine Arginine Histidine Lysinrb Aspartic acid Glutamic acid hmmonia Total ro. of hydrogens i n B

3 8 3 14 16 3

3

ii

6

:3

b

4

20 32 48 28 -

..

..

..

. . . . .

..

8

, .

..

:i

. . . . .

..

..

..

.. ..

..

14

..

..

..

16

..

3

..

, .

, .

ti 4

.. ..

..

..

..

..

..

..

”1 :i‘L IS

28

__

-

66

x

2

!I

-

10

3

38

-

..

55

24.27

protein in solution. Both of these writers use hydrogens by 110 leaving 31 to be accounted for, practically the same analytical data for the kinds and these might well be strongly hydrogen bonded of amiiio acids and numbers of their residues which or buried in the structure. Certainly the availniake up the composition of the protein. Further- ability of labile hydrogens for exchange in the more, Linderstrqhi-Lang considers this protein solid protein differs markedly from the protein to be made up of three chains of residues, giving in solution a t higher temperatures and higher the protein three terminal amino groups and three pH values where the polar water molecules must carboxyl groups. The total possible numbers of relax the molecule. However the observed exexchangeable hydrogens in the protein as given by change of 406 hydrogens is a reasoiiahle check with Linderstr@m-Lang6 are shown in Table I, where, the 450 found by Lang at similar temperatures instead of merely giving the number of hydrogens a t about the isoelectric point of the protein in solufor the amino acid side chains, the labile hydrogen- tion. Adsorption-desorptions carried up to satucontaiiiitig groups are shown. rated vapor pressures of DzO will be done in an atFrom this table it is readily seen that in addi- tempt t o prove whether further exchange occurs tion to 300 hydrogens in the backbone of the pro- as the protein begins to solvate. tein there are 217 possible labile hydrogens in the Using the sorption data at 17 and 27’, ab shown terminal groups and in the various side chains of in Fig. 1 and 3, differential heats of adsorption and the known amino acid residues. Lang6 finds desorption for HzO and DzO were calculated by that theoretically, 547 hydrogens do exchange in the Clausius-Clapeyron method. These heat solution at higher temperatures and at high pH values were then plotted against the amounts of 1-alues, hut u t ordinary room temperatures and sorbed H20 and DzO, expressed in millimoles per at pH of the isoelectric point of 5.25 only ahout 450 gram of protein. The resulting curves appear in of these hydrogens exchange with deuterium. In Fig. L4. The dashed sections of thc rurveh iiidithe present study on the dry lyophilized protein, cate possible lack of precisioii due to slight inacthe mauimiun weight increase is due to an exchange curacies in the sorption values at v c ~ ylow v:~por of $06 hyvdrogci~s per mole of protein having a pressures. It should he pointed out that a iiuml)r.r molcrulor \\ cight of 37,300. I t is thus ohviouy of extra poiiits actually were dctermiitcc-1 for w r h 141 supposedly lahile hydrogens do isotherni at low relative pressure