Salting In and Salting Out

Chapter 12 Salting In and Salting Out

S o m e specific methods of protein purification can now be discussed. Let us start with the effects of various salts on proteins and consider them from both a theoretical a n d a practical point of view. Ammonium sulfate a n d sodium sulfate are often used as precipitating (salt­ ing-out) reagents. T h e technique is simple. We add the salting-out reagent to reach s o m e specific per cent of saturation of the salt. Naturally, different proteins precipitate at different percentages of saturation. Therefore, the liter­ ature includes the expression that a particular protein "was a part of" the 2 0 4 0 % a m m o n i u m sulfate fraction. Translation: First, the investigators brought the protein solution to 2 0 % of saturation and then they removed any precipi­ tate [ 0 - 2 0 % (NFU^SCU fraction] that may have formed; next, they brought the solution u p to 4 0 % saturation and then removed and used the precipitate as the 2 0 - 4 0 % fraction. The removal of the precipitate is usually d o n e by centrifugation. T h e supernatant may b e used to generate additional fractions. Table 12-I shows how many grams of solid a m m o n i u m sulfate must b e a d d e d to 1 liter of a given solution at a particular ammonium sulfate per cent saturation to yield another known per cent saturation. Although these tables d o take the volume correction into account, they are only accurate for a pure w a t e r - a m m o n i u m sulfate solution at 25°C. Solubilities of salts are very tem­ perature dependent; for example, a m m o n i u m sulfate precipitation is usually d o n e at 2 0 - 2 5 ° C because a m m o n i u m sulfate is more soluble at that temper­ ature. Bacterial problems are generally nonexistent at these high salt concen­ trations. A note o n temperature: We often take a protein from an ice bucket, a d d the "correct" concentration of a m m o n i u m sulfate, stir for 1 0 - 1 5 min, and then centrifuge—all at s o m e unspecified temperature. S o m e researchers consist­ ently work at 4°C while using the 25°C table. Sometimes, however, we have 117

TABL E 1 2 - 1 AMMONIU M SULFAT E SATURATIO N TABLE " 118

Fina l concentratio n of ( N H ) S 0 4 , % saturatio n

42

10

20

25

30

Initia l concentratio n of ( N H ) S 0 , 42 4 % saturatio n 0 10 20 25

33

40

50

60

70

75

80

90

100

Solid ( N H ) S 0 t o b e adde d t o 1 liter of solutio n (g)

42 4

56

114

144

176

196

243

313

390

472

561

616

662

767

57

86

118

137

183

251

326

406

449

491

592

694

29

59

78

123

189

262

340

382

424

520

619

30

49

93

158

230

307

348

390

465

583

30 35 40 50 60 70 75

119

80

0

90

19

62

127

198

273

314

356

449

546

43

107

177

252

292

333

426

528

63

132

205

245

285

375

465

66

137

176

214

302

392

69

105

143

227

314

35

72

153

237

36

115

198

77

157 79

Thi s tabl e indicate s th e correc t amoun t of solid ( N H ^ S C ^ (at 25 C ) t o b e adde d t o 1 liter of solutio n t o produc e a desire d chang e in th e pe r cen t saturatio n of ( N H ) S 0 4 . Saturate d ammoniu m sulfat e at 25 C is 4.1 M an d require s 7 6 7 g salt/liter . "Pe r cen t saturation " in thi s tabl e is percentag e of 42 4.1 M. Th e listed value s wer e calculate d fro m table s of pe r cen t salt , specific gravity , an d gram s pe r liter at variou s concentrations .

120

12. Saltin g In an d Saltin g Ou t

critical cuts to make; we may be enriching our solution for o n e of two materials that fractionate at very similar percentages. For example, a 5 3 % a m m o n i u m sulfate cut has b e e n d e e m e d to give the best enrichment of the lower fraction (i.e., the 4 0 - 5 3 % fraction) for the muscle protein troponin. In fact, however, any a m m o n i u m sulfate saturation above 4 9 % would contain some tropomyosin impurity, but our yield is too severely reduced at 4 9 % saturation. In this case temperature control can b e of great importance be­ cause accurate fractionation is required. We should also note that there are two ways of doing these salting-out fractionations, a n d they occasionally yield different results. The first method is easier; we a d d a m m o n i u m sulfate crystals directly into a beaker containing a magnetic stirrer. This creates an a m m o n i u m sulfate gradient moving away from each crystal as the crystals start to dissolve; there is a 1 0 0 % saturated salt solution closer to the crystals. Unfortunately, some proteins do precipitate that should not. Although we can usually resolubilize the proteins, we must be aware that sometimes the high a m m o n i u m sulfate concentration precipitates them irreversibly. The second method of accomplishing this precipitation is to make a 1 0 0 % saturated ( N H ^ S C ^ solution which is then left in contact with excess solid a m m o n i u m sulfate. (Apparently this solution does not supersaturate.) Finally, we a d d the saturated solution slowly with rapid stirring to minimize any gradient. This is the better technique for critical applications. We will examine certain mathematical aspects of salting in and salting out so that we can 1. Develop equations that may b e useful 2. Develop s o m e insight as to the basis of these concepts (both as physical chemists a n d as food scientists) 3. Recognize again that complexities await us It is not sufficient to copy an equation a n d just "plug in" numbers because it is the derivation itself that shows us the underlying assumptions, potentials, and limitations of these processes. The solubility (S) curves for s o m e proteins are plotted in Fig. 12-1 as the log of S/S' versus ionic strength ( S ' is the solubility at zero ionic strength). Thus, the solubility differences at zero ionic strength are essentially normal­ ized. Notice on the far left that protein solubility goes u p as we add salt: This is the salting-in p h e n o m e n o n . As we go to higher concentrations, we notice that proteins stay in solution even at ionic strengths of 3 - 4 M for sodium chloride and potassium chloride. However, the a m m o n i u m sulfate and potassium sulfate curves clearly b e n d at these salt concentrations because proteins are precipitating out of the solution: This p h e n o m e n o n , of course, is salting out.

Saltin g In an d Saltin g Ou t

121

1.4

~*0

1.0

2.0

3.0

4.0

Ioni c strength , μ Fig . 1 2 - 1 . Solubilit y of a protei n (carboxyhemoglobin ) in t h e presenc e of variou s salts . (Take n wit h permissio n fro m Mahler , H . R., an d Cordes , ¯. ˙ . 1 9 6 6 . "Biologica l C h e m i s › try. " Harper , N e w York . A s adapte d fro m Green , A. A. 1 9 3 2 . J. Biol. Chem. 95, 47.)

The difference in solubility between potassium chloride and potassium sulfate obviously suggests that the solubility difference exists between the chlorine a n d the sulfate ions. The Hofmeister or lyotrophic series is a specific way to order ions based on a n u m b e r of their properties; note that o n e end of the list includes the preferred salting-out compounds. Interestingly, those ions involved in salting out favor the helix configuration and "native" conforma­ tion, that is, m o r e compact structures. Certain general effects occur regardless of which specific ions are present. We try to account for these properties in terms of ionic strength (J = iEmjZf ) which is d u e to the charged sphere that all ions have (see Table 12-11). Specific ion effects are those superimposed on this general effect. Let us return to the salting in at low ionic strength. First, there is the equilibrium solubility product which relates to the salting in of a protein thus:

+

2

+

K sp = ( α Α) (+α Β- ) = γ + ( Α ) + γ_(Β") = γ ±( Α ) ( Β " )

(12-1)

122

12. Saltin g In an d Saltin g Ou t TABL E IONI C STRENGT H

12-11 CONSIDERATION S

Measur e of electrostati c interactio n betwee n ionic charges :

m = molalit y x Cj = mola r concentratio n Mola r concentration : C = - , f* * 1Λ+ (mM J XM æ = densit y of solutio n (if m M / 1 0 0 is smal l an d æ p ) r 0 M = molecula r weigh t r m = molalit y In dilut e solutions : C = po m po = densit y of solven t Propertie s dependen t upo n ioni c strength : Activit y coefficient of electrolyte s Solubilit y of sparingl y solubl e salt s Rate s of ioni c reaction s Primar y kineti c salt effect: (a + b ^ c) S a m e sign of charge rat e constan t increase s a s / increase s Opposit e sign of charge rat e constan t decrease s a s J increase s N o sign n o chang e in rat e constan t Iner t salt use d t o minimiz e effect: NaC l

The solubility product is a true thermodynamic term a n d is therefore a con­ stant under defined conditions; it is related to the activity of the positive ion and the activity of the negative ion. The concentration of proteins is used as a first approximation because we know very little about the protein activity. The solubility product is then not a true thermodynamic value. If (a A+) (as-) is greater than K s ,pthe sample precipitates. The data for activity coefficients of salts (Fig. 12-2) are plotted as molarity versus y±. For s o m e salts, y± drops sharply with concentration creating situa­ tions in which the salt naturally solubilizes itself. Notice that the curve for sucrose never goes down: It does not solubilize itself. By means of the Deb y e - H u c k e l theory (see below), we can calculate the decrease in y for the region of the curve slightly to the right of the y axis. In this region, salts are essentially still behaving "ideally," that is, n o i o n - i o n interactions are occur­ ring. This point is literally comparable to a drop in the ocean: This is an extremely dilute solution which exists in theory only. Nevertheless, the Deb y e - H u c k e l theory is a valuable conceptual tool that helps us understand the

123

Salting In and Salting Out

Fig. 12-2. Mean molal activity coefficients of electrolytes. (Taken with permission from Moore, W. J. 1962. "Physical Chemistry," 3rd ed. Prentice-Hall, Englewood Cliffs, New Jersey.)

effect(s) of the activity coefficient and permits us to make certain calculations _1

i

°S У =

1.81 x IQ* D3/2*p/3

z

iz2

V

r

M

(I 2 " 2 )

where D = dielectric constant for the medium, T = temperature, fi = ionic strength, and Z\, Z2 = ion charges. (Unfortunately, the symbols used for a particular quantity are not always consistent within the literature. Ionic strength is a good example: д, Г/2, /.) If we increase the ionic strength or the charge, we get a larger -log у term; assuming Ksp does not change, this leads to an increase in solubility. Proteins generally act as multivalent electrolytes. The dielectric constant of a solution is a measure of the force of interaction between two charged particles in this medium. If particle 1 and particle 2 are of opposite signs, the two particles have an attraction that is proportional to the product of their charges. The force of attraction between these two particles decreases by the square of the distance between them. This, however, is only true if the two particles are in a vacuum. If they are in a real fluid, we need an

124

12. Saltin g In an d Saltin g Ou t

adjustment factor because there is an additional barrier to the strength of the interaction. This interaction factor is the inverse of the dielectric constant; as D increases, the protective barrier is greater. Water has a particularly high dielectric constant, enabling it to block charge interactions easily. Consequently, it is a desirable solvent to use for charged materials because it prevents opposite charges from coming together and allows pairs of the same charge to get relatively near each other. (Although very complex, the proteins can still be thought of as being essentially a salt. ) The D e b y e - H u c k e l calculations show the effect of increasing salt concen­ tration on the ions themselves. However, the addition of salt also has a similar effect on the activity coefficient of other ions in solution, and it is this latter interaction of salts that leads to the salting in of proteins. These calculations, unlike the data of Fig. 12-2, ignore the individual ion's specific properties. Figure 12-3 shows the salting-in effect of p H and ionic strength for β-lactoglobulin, a protein whose minimum solubility is at its isoelectric point. 3.2

Fig . 12-3. Solubilit y of -lactoglobulin . N o t i c e bot h th e c h a n g e in t h e p H m i n i m u m an d t h e d e c r e a s e in t h e solubilit y a s N a C l concentratio n decreases . (Take n wit h permissio n fro m Mahler , H . R., an d Cordes , ¯. ˙ . 1 9 6 6 . "Biologica l Chemistry. " Harper , N e w York . A s adapte d fro m Fox , S., an d Foster , J . S. 1957. "Introductio n t o Protei n Chemistry. " Wiley , N e w York. )

125

Saltin g In an d Saltin g Ou t

As we increase the salt concentration, we get a marked increase in the a m o u n t of material soluble at a given pH. The shift of the isoelectric point with the change in salt concentration is especially noteworthy. Other solvents have lower dielectric constants than that of water. Organic solvents decrease the dielectric constant, which means there is less protection from charge interactions. Initially, the protein tends to swell and unfold. Charge separations that exist at a certain distance in water must move further apart in an organic solvent. For proteins (with their complex conformations) this causes a dissociation and unfolding, permitting the hydrophobic groups to interact just as easily with the solvent as they can with themselves. (In water, they are trying to "get away" from the water.) The thermodynamic advantages of reacting with "self" as opposed to solvent water are n o longer present. The forces of electrostatic charge interactions and the forces of hy­ drophobic interactions are both showing a tendency for proteins to unfold in organic solvents. Only the formation of hydrogen bonds seems to favor an increase in the order or stability because hydrogen b o n d formation tends to remove s o m e of the effects of charge. With highly organic (nonpolar) solvents, it has b e e n postulated that the outside of the protein becomes hydrophobic. Insoluble particles are obtained on drying granular proteins from these solvents. These proteins may b e useful nutritionally but are not particularly "functional." Normal fish protein concen­ trate is an example of a material prepared using organic solvents. Salting out may occur if we run a material through an ionic exchange column for desalting. We may lose the salting-in effect and instead get an insoluble material that sticks onto the gel a n d plugs u p the column. W h e n + + desalting, however, we must deal with the counterions n e e d e d to maintain charge neutrality. Desalting is an " e x c h a n g e " of other ions for N a or H a n d / o r CI" or O H " ; this is important to remember when we use ion exchang­ ers for water "purification" purposes. Salting out yields a lot of empirical data. Figure 12-4 shows that we can get an extreme change in protein solubility over a very small range of ionic strengths [(NH4)2S04 concentrations]. We can also see tremendous differ­ ences in ionic strength ranges for the salting out of different proteins. This can be very helpful. For example, we have n o trouble separating hemoglobin and myoglobin: At an ionic strength of 5.7, myoglobin is still soluble and h e m o ­ globin is not. Notice too that the slopes of most of these curves are approxi­ mately the same. These observations can be represented by the empirical formula: logS = j 3 - K i / x where K's = salting-out constant, S = solubility, and β = a protein constant.

126

12. Saltin g In an d Saltin g Ou t

Ioni c strength , μ Fig . 12-4. Th e solubilit y of protein s in a m m o n i u m sulfat e solutions . (Take n wit h permis › sion fro m Mahler , H . R. , an d Cordes , ¯. ˙ . 1 9 6 6 . "Biologica l Chemistry. " Harper , N e w York . A s adapte d from Cohn , E. , an d Edsall , J . T. 1 9 4 3 . "Proteins , A m i n o Acids , an d Peptides. " A c a d e m i c Press , N e w York. )

This is a linear equation. The slope (-K's) of the line is a function of the specific salt being added; the intercept (β) is a function of the individual proteins a n d should not change as the precipitating salts are changed. Thus, the slope for a m m o n i u m sulfate, for example, could be calculated by averag­ ing all of the a m m o n i u m sulfate slopes. We could calculate β for any protein by averaging the intercepts of different salts. Salting out might b e explained thus: When numerous salt ions are in solution, they must b e neutralized or shielded from other charges. This is d o n e by the water molecules. However, if enough water molecules are tied u p with the salts, there are not enough water molecules left for the proteins. The protein molecules consequently interact with themselves and this leads to precipitation. Regions of opposite charge presumably come together; hydro­ phobic bonds may also b e formed. Another qualitative if somewhat more sophisticated explanation would incorporate the fact that water is an excep­ tional hydrogen bonding agent (2 Η b o n d s / 1 8 daltons). Because of its bonds, water has a very o p e n structure; it is a lattice of so-called holes or spaces which are more n u m e r o u s in ice than in water (which is why ice floats on water). Salts fill many of these holes and break the organized water structure, causing it to collapse. Ultimately, there are no holes or spaces for the protein. Procedures that shown an ionic strength dependence should usually be expressed in terms of the specific salt added. However, we can only speak of

Saltin g In an d Saltin g Ou t

127

an ionic strength effect if more than o n e salt is used a n d the multiple results are identical. H o w d o we explain the charge contributions to the ionic strength of the protein solution itself? As a large multicharged molecule, the protein's contri­ bution to the properties of the solution is not clear. It seems that there are two extreme hypotheses. The first is that the particle acts as a single material having a net charge. We will use this assumption when we calculate the Donnan equilibrium in the next chapter (Chapter 13). Mathematically, this m e a n s that the protein concentration is simply multiplied by the square of the net charge (see Table 12-111). The second hypothesis is that each of the charges acts as a point charge. In this case, we n e e d to know the n u m b e r of plus charges and the n u m b e r of negative charges for the protein under specified solvent conditions. As the charge in each case is one, the ionic strength contribution will b e the n u m b e r of charges multiplied by the protein concentration. If any small ions bind to the protein, then a correction must b e m a d e to the theoretical charges, for example, the charge calculated from the amino acid analysis. S o m e w h e r e between these two hypotheses lies a third possibility, that there are areas of charges that act as a point charge, that is, charge clusters (see Table 12-111). Sometimes we want to study changes such as myosin solubility that are sensitive to small ionic strength changes. Insoluble myosin becomes soluble as the molarity increases from 0.25 to 0.35 M NaCl. But if we want to predict the salting-in range for other polyvalent ions, we must remember that the actual ionic strength, properly calculated (including the protein's contribu­ tion), may b e c o m e m o r e important. A study of myosin filament solubility as a function of protein concentration could tell us something about the protein's contribution to ionic strength regardless of protein concentration or the in­ volvement of any specific ion effects. We may also find that filaments form only after the protein concentration is above the critical micelle concentration for this protein association. Solubility measurements are a commonly measured functionality property. The food scientist would like to have a standardized solubility test that can b e used to compare different manufacturers' products or ingredients in a m a n n e r that is fair to all concerned parties. But we should note from the theoretical discussion a b o v e that manufacturers could increase the apparent solubility of their proteins simply by increasing the a m o u n t of salt in a product. In practical situations, product d e m a n d s may require solubilization of a protein at a partic­ ular pH, but advertising d e m a n d s might favor a p H that is far from the point of minimum solubility. (The point of minimum solubility and the p H n e e d e d in many foods are both often in the p H 3 - 6 range. ) A more general discus­ sion of food protein functionality appears in Chapter 27.

128

12. Saltin g In an d Saltin g Ou t TABL E 12-111 CALCULATIN G TH E IONI C STRENGT H OF PROTEIN S

+ 1 Na

+ 1 Na

+ + +

3 CI

+ + +

3 CI " Counterion s (CI ) ar e ionize d an d

+ 1 Na

-

1 Na+

-

+ + + + +

monovalen t

3 CI

3 CI

Protei n 1 mo l of protei n with counterions . Th e protei n ha s 4 negativ e charge s an d 12 positiv e charge s in 4 separat e clusters . Assumption : Net charge :

+8

2

ˇ ˘ 2 -~=[(1)S }

+ [(8)1 ] = 3 6 M

+

(CI ) Eac h charg e treate d individually :

1 2 , 4~

2

2

yr

7

= [(12)1 J

2

2

+ [(12)1 ] + [(4)1 ] + [(4)1 ] = 16 M (CI )

+

(CI )

Cluster s with ne t charge : 4 x 2 -

ˇ ˘ 2 ~ = [(4)2 ] + [(8)1] = 12 M (CI )

or

2

2

2

[(4)2 ] + [(12)1 ] + ( 4 ) 1 = 16 M (CI )

(CI )

Most solubility tests include mixing a relatively insoluble material with the solvent (usually water) for u p to 1 5 - 2 0 min. Clearly, this may not be enough time to obtain maximum solubility. We should also at least try to mix the solutions in the s a m e m a n n e r each time for the sake of consistency. We then centrifuge or filter the solution to remove the insoluble material and perform a protein determination on the supernatant. (Remember that thermodynamics

129

Saltin g In an d Saltin g Ou t

describes the conditions that exist at equilibrium, kinetics quantifies the time required to reach equilibrium.) In an effort to b e more precise, we can measure the p H of the solution and adjust it to the actual p H being reported, remembering that mixing a dry powder with a solution of known p H may not yield a solution with the same final pH. Unfortunately, this process changes the salt concentration and the volume. The results may also d e p e n d on the purity of the water we use; tap water in industrial situations may have more salt than distilled water. We may get specific ion effects on the proteins from the magnesium and calcium ions of hard water. Problems like these sometimes make it difficult to apply lab data to a particular industrial situation. If we really want critical comparative data, we should probably spend 1 - 2 days dialyzing all materials to consistent and identical conditions (see Chapter 13). A frequently used research procedure (used in the solubility test described above) is to change the p H of a solution. W e add either an acid or a base into the protein solution with stirring. We must b e aware of potential problems, however. If the protein solution is very viscous, the addition of even a dilute acid will yield a solution of "jelly b e a n s . " O n e example is the addition of a molar solution of HC1 to myosin. (A stock solution of HC1 is 11.6 M and the chemist's "dilute" normally implies 6 M.) The acid is strong enough to be a nucleating center for precipitation; clumps of acid precipitated protein form around the core of HC1, a n d the precipitated myosin is functionally inactive. We can change the final p H using a more dilute solution [0.1 M HC1 or 1.0 M CH3COOH (acetic acid)]. However, this procedure may cause a greater change in volume. In the case of CH3COOH, this also means we are adding the acetate ion. But if we are going to lyophilize the sample, the excess acetate is volatile. (Remember that proteins are good buffers. Therefore, the a m o u n t of solution n e e d e d to adjust the p H is often more than anticipated. This adjustment tends to shift both the solution volume and the ionic strength.) PROBLEM SET Starting with 5 0 0 ml of a protein solution at 25°C, we add 2 0 0 ml of saturated ( N H 4) 2S 0 4 solution. The final volume is 6 9 0 ml. We centrifuge the sample a n d recover 6 7 0 ml of supernatant. To this supernatant we add 100 g of solid (NH4>2S04. After dissolving the salt, the solution is centrifuged and the precipitate then represents the protein obtained between and % a m m o n i u m sulfate saturation.