Thiophilic adsorption of immunoglobulins—Analysis of conditions optimal for selective immobilization and purification

ANALYTICAL

BIOCHEMISTRY

159, 2 17-226 (1986)

Thiophilic Adsorption of lmmunoglobulins -Analysis of Conditions for Selective Immobilization and Purification T. WILLIAM HUTCHENS’ Institute

of Biochemistry,

University

of Uppsala,

Uppsala

Optimal

AND JERKER PORATH* Biomedical

Center,

Box 576. S-751 23 Uppsala,

Sweden

Received March 28, 1986 Immunoglobulins have been selected by their general affinity for adjacent sulfone-thioether sulfur groups as a useful model system for the characterization of thiophilic interaction chromatography. Mercaptoethanol coupled to divinylsulfone-activated agarose (thiophilic or T-gel) provided an affinity matrix for the efficient and reversible immobilization of the immunoglobulins. The adsorption/desorption process was investigated as a function of protein concentration, temperature, flow rate, and pH in different concentrations of ammonium sulfate. Immobilization of these proteins was (as a function of pH) found to be both dependent and independent of the adsorption-promoting effectsof water-structure-forming salts. Buffer conditions are recommended for the selective adsorption of immunoglobulins from unfractionated human serum. These results indicate that thiophilic interaction chromatography provides a new and effective alternative for the immobilization and purification of immunoglobulins and other proteins under conditions known to preserve structure and biological activity. 0 1986 Academic Press. Inc. KEY WORDS: thiophilic gel; adsorption; immunoglobulins; protein immobilization; protein purification; affinity chromatography.

Techniques allowing the effective immobilization of ligands for protein adsorption (1,2) continue in their development. As a result, affinity chromatography methodologies (3,4) have also evolved. However, with each new immobilizing matrix the possibility for multiple types of interaction mechanisms, often with unintended proteins, has been revealed. Analyses of these phenomena have recently led to the discovery of a previously unrecognized type of protein-ligand interaction (5). This new interaction is termed thiophilic since it is distinguished by proteins which recognize, through as yet unknown mechanisms, a sulfone group in close proximity to a thioether (5). The characteristic ligand structure is -SO*-CH2-CH2-X-R. ’ Permanent address: Department of Obstetrics and Gynecology, Baylor College of Medicine and the Reproductive Research Lab, St. Luke’s Episcopal Hospital, Houston, Texas 77030. * To whom correspondence should be addressed.

If a thiol compound, RSH, is coupled via an oxirane group the resulting gel derivative will not be thiophilic. To exhibit thiophilicity, the adsorbent must thus have a sulfone group in the ligand. The structural requirement for X is less critical, but X must be an atom with a lone electron pair. Certain aliphatic amines will produce thiophilic adsorbents when coupled to vinyl-sulfone-agarose (but not to oxirane-agarose), but the thiophilicity is much weaker than for the T-gel3 (5). The T-gel used in this study can be represented as agarose-0-CH2-CH2-SO*-CH2-CH2-SCH2-CH2-OH. A similar gel with penultimate hydroxyl has almost the same capacity as the T-gel. This latter ligand is hydrophilic rather than hydrophobic. Thiophilic adsorption differs from hydrophobic interaction in some very important re’ Abbreviations used: T-gel, thiophilic gel; Hepes, 4-2hydroxyethyl- I -piperazine-ethanesulfonic acid.

217

0003-2697186 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form resewed.

218

HUTCHENS

spects. The latter is strongly promoted by high concentration of sodium chloride, whereas thiophilic adsorption is weakened. The proteins adsorbed to a hydrophobic gel such as octyl-agarose are of different nature from those thiophilically adsorbed. For example, albumin among the serum proteins is not at all adsorbed to a T-gel whereas it is the major protein interacting with a hydrophobic gel. The reverse is true for the immunoglobulins. The selectivity in adsorption with respect to immunoglobulin classes and subclasses has not been studied but it is evident from available preliminary experimental data that the “class spectrum” is broader than for Protein A. A preliminary report describing tandem arrangements of thiophilic and hydrophobic interaction columns to differentially adsorb serum proteins has shown the potential utility of thiophilic gels as specific adsorbents for immunoglobulins. The results presented here are derived from investigations aimed towards the effective utilization of thiophilic-interaction chromatography for the selective immobilization of immunoglobulins. MATERIALS

The T-gel was synthesized as described previously (5). Spherical beads of agarose (6%) were activated with divinylsulfone to couple mercaptoethanol. The ligand concentration estimated to be approximately 1 mmol g-’ dry gel. Only unused T-gel was used for each experiment described here. Normal human serum immunoglobulins (y-globulins) were generously supplied by KabiVitrum, Stockholm, Sweden. All buffers and reagents were of standard quality. The buffer system used for the pH-dependence studies (Fig. 6) consisted of a 0.2 M solution of the following constituents: citric acid, sodium phosphate, 4-morpholine-ethane-sulfonic acid, 4-hydroxyethyl-piperazine-ethanesulfonic acid (Hepes), and glycine. Chromatographic details are provided in the figure legends.

AND

PORATH

RESULTS

Adsorption Isotherms The fundamental relationship between sample protein concentration and column adsorption capacity was determined both as a practical guide for efficient column use and to better orient future efforts towards the development of displacement chromatography with the thiophilic gel. The thiophilic adsorption isotherms shown in Fig. 1 reveal an early and sharp transition for the adsorption of y-globulins. Under these conditions the shape of the isotherms and the apparent column capacities are nearly the same at 4 and 20°C. The adsorption capacity of the T-gel was determined by replicate frontal analyses at each of the protein concentrations (0.17 to 10.6 mg/ml) shown. The individual frontal analyses consistantly yielded continuous, sigmoidal curves with little or no evidence for the presence of any but one single interactive sample component. Upon apparent saturation with protein, the columns were washed with column and sample equilibration buffer until near baseline levels (by absorbance at 254 nm) of protein were present in the column eluate. The column (T-gel) protein-binding capacity was then determined in two ways. The amount of sample required to reach 50% column saturation was calculated and compared to the actual amount of sample protein retained by the column after extensive washing to remove unbound protein. Good agreement between these two methods was observed using protein concentrations up to approximately 5 mg/ml. At higher protein concentrations (e.g., 10.6 mg/ ml) correlation of values obtained by these two means was inconsistant from nine separate experiments. Reasons for this disparity will be discussed. However, the adsorption capacity of approximately 65-70 mg protein/g (suction dried) of T-gel agrees with values obtained by direct titrations of the T-gel with increasing concentrations of immunoglobulin. Analysis of this same data by the method of Scatchard (9) suggests an affinity dissociation constant (&) of l-2 X 10e7 M (data not shown).

THIOPHILIC

I

2

4

ADSORPTION

6 Sample

6

10 protein

219

OF IMMUNOGLOBULINS

I concentration

2

4

6

6

10

(mglml)

FIG. 1. Thiophilic adsorption isotherms of y-globulins at 4°C and 20°C. The adsorption capacity was determined by frontal analysis for each of six protein concentrations ranging from 0.17 to 10.6 mg/ml. Chromatography was performed using jacketed Pharmacia C- 10 (1 .O id.) columns containing 1.OOg of suction-dried T-gel (1.35 ml bed volume) at a flow rate of 15.8 or 16.5 cm/h. Sample and column equilibration buffer was 20 mM Hepes (pH 7.5) containing 10% (100 g/liter) ammonium sulfate. Column operating temperatures (4 and 20°C) were maintained using a constant-temperature circulating water bath. The temperature of both reservoir and column jacket outlets were continuously monitored. Sample elution profiles were obtained using flow-through UV (254 nm) absorbance monitors and strip chart recorders. Closed circles represent amount of protein retained by column at apparent saturation and was determined after extensive washing with sample and column equilibration buffer by calculating the difference between total sample amount added and unadsorbed protein collected in wash-through fractions. Open circles represent amount of protein added to reach 50% of the eluate absorbance maximum at apparent column saturation.

Adsorption Capacity and Flow Rate It was of practical interest to know if sample application and eluent flow rates significantly influenced thiophilic adsorption capacity for y-globulins. Because of the rigidity conferred upon agarose by crosslinking with divinylsulfone ( 10) flow rates up to 65 cm/h (5 1 ml/h) were investigated. Furthermore, the dependence of adsorption capacity on flow rate during chromatography was examined at two different protein concentrations (2.5 and 10.6 mg/ml) to better evaluate apparent complications due to possible protein-protein interactions. As shown in Fig. 2, the adsorption capacity is effectively decreased at the elevated flow rates. This effect was less pronounced when the lower protein concentration was used even though protein concentrations of both 2.5 and 10.6 mg/ml appear in the linear plateau region of the adsorption isotherm under these same conditions (Fig. 1). As described for Fig. 1, the column adsorption capacity for

each experiment was determined by two means using frontal analysis. The amount of sample (volume X absorbance units per milliliter at 280 nm) required to reach 50% column saturation was calculated from the frontal curve and compared to the amount of sample actually retained by the column after removal of unbound protein (amount of sample applied less unbound protein measured after extensive washing). The differences between results obtained by these two means appear to be more constant at each flow rate investigated and are clearly more pronounced at the higher protein concentrations. These results are consistant with those presented in Fig. 1. Salt-Promoted Ammonium

Adsorption with Sulfate

The adsorption of serum proteins to the Tgel has been shown in preliminary experiments to be dependent upon the presence of a waterstructure-forming salt (5). The lyotropic salts

220

HUTCHENS

AND POBATH

I

1

1

20

20 Flow

,a::

(~rn.h-;~

1

c

40

Flow

rate

60

(cm-h-‘)

FIG. 2. Effects of sample and eluant flow rate on thiophilic adsorption of y-globulins. Chromatography was performed at 4 (A) and 2O’C (B) as described for Fig. 1. Adsorption capacity was determined by frontal analyses using protein concentrations of 2.5 and 10.6 mg/ml at flow rates from 6.8 to 64 cm/h developed using peristaltic pumps. The results are expressed as the mean values obtained from two to five separate experiments at each flow rate. Closed and open circles represent values obtained by the two different means as described in the text and for Fig. 1.

are arranged in the Hofmeister series with ammonium ions and sulfate ions both at or near the same end of the spectrum. Ammonium sulfate was thus selected as a logical salt for these studies. Furthermore, its wide-spread use as a protein precipitant during early phases of protein purification schemes could be complementary to its ability to promote thiophilic adsorption. Figure 3 demonstrates that the yglobulin proteins are adsorbed to the T-gel in a manner which can be both independent as well as dependent upon the presence and concentration of ammonium sulfate. Under these conditions only 3-4% ammonium sulfate was required to reach 50% of maximal binding to the T-gel. The level of y-globulin adsorption to the T-gel in the absence of ammonium sulfate (20 tnM Hepes only) was significant and appeared in contrast to results presented earlier (5). We thus investigated the possibility that ionic strength of the buffer constituent alone could effect immunoglobulin adsorption. These results are presented in Fig. 4. In the absence of ammonium sulfate (or other waterstructure-forming salts), increasing concentrations of sodium chloride significantly re-

duced the extent of y-globulin adsorption in the absence of ammonium sulfate. As shown in Fig. 4, the adsorption of y-globulin in 0.1 M Tris-HCl alone (see Ref. (5)) was less than half of that observed using 20 mM Hepes buffer at the same pH. We therefore evaluated the effects that inclusion of 0.5 NaCl might have on the ammonium sulfate-dependent thiophilic adsorption of y-globulins. As shown in Fig. 5, the inclusion of 0.5 M sodium chloride did not reduce the thiophilic adsorption promoted by 10% ammonium sulfate. As expected from results shown in Fig. 4, however, 0.5 M sodium chloride did greatly improve (by twofold) the desorption efficiency (generally 85-90% recovery) obtained by removal of the ammonium sulfate. Importantly, similar desorption efficiency could be achieved by introducing the sodium chloride subsequent to protein adsorption in its absence. Table 1 shows that other salts may also be used effectively to promote the thiophilic adsorption of immunoglobulins. There may be an inverse relationship between the degree of adsorption efficiency and recovery. These studies, however, need to be extended using

THIOPHILIC 0.5M

P

a b

m 22

ADSORPTION

221

OF IMMUNOGLOBULINS PerC*“t

K2S04

20

40

of iota1 60

60

100

t 83%

60

: .c 60 al z 'a 40 4z 100

Bound E,“,ed, ElutedP

1 iBound t cpy~E,u,ed, Eluted2 2

:,/-

Bound

i

0

2

4

Percent

6

ammonium

6 10 sulfate

FIG. 3. Effects of ammonium sulfate concentration on thiophilic adsorption of y-globulins. Protein sample (0.5 mg/ml) and column equilibration buffers consisted of 20 mM Hepes (pH 7.5) containing O-IO% (w/v) ammonium sulfate. Small diameter columns (0.5-cm i.d.) were packed with 0.405 g of suction-dried T-gel and eluted (20°C) at approximately I ml/min. The column sample load at each concentration of ammonium sulfate was 2.0 mg (4 ml). Eluted protein was determined by its absorbance at 280 nm. Replicate experiments are shown. The arrow at 0.5 M (6.6%) ammonium sulfate represents the level of yglobulin adsorption achieved using an equivalent concentration (0.5 M) of potassium sulfate.

highly purified protein homogeneous in structure. The tendency of other purified model proteins to be adsorbed by the T-gel in the absence

87%El”tedl El”ted2

---_-------__-.------------------+ ) -0 5M NaCl _- __--------------

e-O5MNaC,

-------------------I -0 5M NaCl

+0.5MNaCl

- ___...._---------------------------

-0 +lJ.5MtqaC, +0.5M NaCl

4 -0

!jMNaC,

- 0.5M NaCl +0.5MNaCl

FIG. 5. Combined effects of sodium chloride and ammonium sulfate on thiophilic adsorption and desorption efficiency. Sample and column equilibration buffers consisted of 20 mM Hepes (pH 7.5) containing 10% ammonium sulfate in the presence and absence of 0.5 M sodium chloride as indicated. Elution buffers were 20 mM Hepes (pH 7.5) with and without 0.5 M sodium chloride as shown. Chromatography was performed (20°C) as described for Fig. 3.

of water-structure-forming salts is generally low regardless of isoelectric point (surface charge), size, or relative hydrophobicity. These results are to be presented elsewhere (11). pH Dependenceof Thiophilic Adsorption

In order to take full advantage of thiophilic adsorption for the immobilization and/or purification of antibodies, it is important to know the extent by which relative extremes in pH effect both adsorption and desorption. The full TABLE 1 SALT-DEPENDENT PROMOTION OFTHIOPHILIC ADSORPTIONOF~MMUNOGLOBULINS: A COMPARISONOFSALTSEFFECTS

a

I 0

* . 01 02 03 Sodturn chloride

' 04 (Ml

. 05

FIG. 4. Effect of sodium chloride concentration on the interaction of -y-globulins with the thiophilic gel in the absence of ammonium sulfate. Sample and column equilibration buffers consisted of 20 mh+ Hepes (pH 7.5) containing O-O.5 M sodium chloride. Chromatography was performed (20°C) as described for Fig. 3. The arrow indicates the level of adsorption achieved in the presence of 0. I M Tris-HCI (pH 7.6) alone.

Salt” WH.XQ Mg SO, KS%

Na2S04

2

Percentage bound 74.1 78.5 83.5 92.2

Recovery 91.2 91.0 87.9 83.2

Note. Immunoglobulins (0.5 mg/ml) sample volume: ml. p 0.5M salt in 20 mM Hepes (pH 7.5) at 20°C.

222

HUTCHENS

range from pH 3 to 9 is important in this regard because of the useful effects that pH 3 and 9 can have on the perturbation of antigenantibody complex formation. A single buffering system was prepared for these studies with sufficient buffering capacity required for this entire range of pH values. This was necessary to help avoid ambiguities regarding the potential differential effects of various separate buffers on thiophilic adsorption potential. When the concentration of ammonium sulfate was enough ( 100 g/liter) to promote complete (97-99%) thiophilic adsorption of the y-globulins, as shown in Fig. 6A, no pH effects were obvious during adsorption. However, the percentage of bound protein desorbed (recovered) upon removal of ammonium sulfate decreased dramatically with increasing acidity. At pH 3 less than 2% of the bound protein was routinely desorbed after washing with over 10 column volumes of buffer. In this regard sim-

A10%(NH4)2S04 100

S 5%(Ntf4)2S04

C O%(NH4)2S04

AND PORATH

ilar results were obtained using lower concentrations of ammonium sulfate. However, by promoting only partial adsorption using 5% ammonium sulfate, significant pH effects on the adsorption process were revealed. The point of lowest adsorption efficiency at pH 5 may reflect the individual contribution of two separate adsorption mechanisms. This was investigated by examination of pH effects on the adsorption process in the absence of any waterstructure-forming salt. The second set of results shown in Fig. 6B indeed suggests that the nearly quantitative adsorption observed at pH 3 could also be entirely independent of ammonium sulfate. The magnitude of this adsorption phenomenon was decreased steadily as a function of increasing pH. In agreement with results shown in Fig. 6A, protein desorption in both the presence and absence of 5% ammonium sulfate increased with increasing pH. Maximizing Desorption Eflciency from Thiophilic Gel

t

t

I... 3579

,

t

16...

3579 pi-f of

1

I;;;, buffer

FIG. 6. pH dependence of thiophilic adsorption/desorption of -y-globulins. Sample (0.5 mgjml) and column equilibration buffer constituents were the same at each pH (see Materials). Columns (OS-cm i.d.) were packed with 0.205 g of suction-dried T-gel for a sample load of 2.0 ml. Chromatography was performed (20°C) as described for Fig. 3. Pane1 A shows the pH independence of the thiophilic adsorption promoted by 10% (w/v) ammonium sulfate. Panel B reveals the pH dependence of adsorption in the presence of only 5% ammonium sulfate. Panel C indicates the pH dependence of adsorption observed in the absence of ammonium sulfate. In each panel, closed circles represent the percentage of protein sample adsorbed. The open circles indicate the percentage of adsorbed protein eluted upon removal of ammonium sulfate from the eluent.

Efficiently utilizing thiophilic adsorption, especially with precious antibodies, requires good recovery regardless of adsorption conditions. Either inclusion or subsequent introduction of 0.5 M sodium chloride generally results in 85-90% recovery of adsorbed immunoglobulins at pH 7.5. Figure 7 shows that up to 85-90% recovery of bound protein can be expected, even after adsorption in acidic buffers, by pH neutralization and inclusion of 0.5 M sodium chloride. Most remaining proteins can be eluted by desorption with 30-60% (v/v) ethylene glycol. It should be noted that the immunoglobulin preparation used for these studies, while providing the necessary quantities for characterization of the T-gel, was not completely homogeneous. After gel filtration of the immunoglobulins on Sephacryl S200 3-4 minor staining protein bands (in addition to the heavy and light chains) were still evident by acrylamide gel electrophoresis under denaturing conditions. Similar analyses of

THIOPHILIC

ADSORPTION

0%

(NH&O4

3

4

5

lO%(NH&

5 %(“H,), T,

6

3

223

OF IMMUNDGLOBULINS

4

5

6

3

4

SO4

5

6

PI-I Of buffer

FIG. 7. Elution of y-globulins adsorbed to the thiophilic gel at acidic pH values. Sample and column equilibration buffers were as described for Fig. 6. The open squares represent the percentage of sample adsorbed. The following symbols indicate the percentage of adsorbed protein eluted by sequential washing with: A, pH buffer (pH 3-6) containing no ammonium sulfate; 0, pH buffer (pH 3-6) containing 0.5 M sodium chloride; 0, 20 mM Hepes (pH 7.5) containing 0.5 M sodium chloride; n , 20 mM Hepes (pH 7.5) containing 60% (v/v) ethylene glycol. The asterisks show percentages of sample recovered.

the immunoglobulin proteins desorbed from the T-gel showed no qualitative differences relative to unfractionated starting material (data not shown). Selective Adsorption of Immunoglobulins from Human Serum The recent initial report on thiophilic adsorption (5) from this laboratory described the use of tandem hydrophobic and thiophilic interaction columns to distinguish their relative adsorption capacities and selectivities for human serum proteins. The principal buffer used for those studies was 0.1 M Tris-HCl (pH 7.6) containing 0.5 M potassium sulfate. As shown in Fig. 3, the thiophilic adsorption of purified y-globulins was nearly the same using 0.5 M potassium or ammonium sulfate in 20 mM Hepes buffer at pH 7.5. However, it remained of interest to know whether the optimal experimental conditions suggested by our current investigations with purified immunoglobulins would result in an increase or decrease in the selectivity of the thiophilic gel

for other serum proteins. The following buffer was chosen based on the present studies to potentially provide both maximum adsorption and desorption of serum immunoglobulins: 20 mrvr Hepes (pH 8.0) containing 7.5% (w/v) ammonium sulfate and 0.5 M sodium chloride. The thiophilic adsorption chromatography of unfractionated human serum proteins was performed in parallel using both the new buffer just outlined and the buffer system used previously (5). The results of this comparison are shown in Fig. 8. The main difference appears to be an increase in the absolute selectivity for immunoglobulins by the T-gel when the new buffer system is used. The quantity of a*-macroglobulin adsorbed appears markedly reduced. Recovery of total proteins in these experiments was similar at 88-92%. In separate studies, individual columns of T-gel have been repeatedly used for serum protein fractionations after regeneration by washing with 95% ethanol. No change in adsorption capacity or selectivity has been noted after more than 30 uses (unpublished observations).

224

HUTCHENS

AND PORATH

A.

B.

I

k 2 t

20

60

40

80 Fraction

100

20

3

45

60

number

s 12

40

1

2

3

S

80

4

100

5

6

6

FIG. 8. Conditions for the selective tbiophilic adsorption of immunoglobulins from human serum. Aliquots of whole serum were dialyzed against column equilibration buffers overnight at 4-6°C. Columns (I.O-cm i.d.) were packed with 1.O g of suction-dried T-gel (I .35 ml bed volume). Chromatography was performed (20°C) essentially as described for Fig. 1. After loading 1.85-2.0 ml of sample (150 absorbance units at 280 nm) unbound proteins were eluted with column equilibration buffers which were (A) 0.1 M Tris-HCl (pH 7.6) containing 0.5 M potassium sulfate or (B) 20 mM Hepes (pH 7.5) 7.5% (0.568 M) ammonium sulfate, and 0.5 M NaCl. Elution of adsorbed proteins was initiated at arrow 1 by removal of (A) potassium sulfate or (B) ammonium sulfate and at arrow 2 by introduction of 60% (v/v) ethylene glycol. Under these conditions, recovery is typically 85-92%. The chromatograms are shown at top. (Bottom-left) Electrophoresis on polyacrylamide gradient gels (PAA 4/30 obtained from Pharmacia, Uppsala, Sweden) of the peak fractions obtained after the thiophilic adsorption chromatography using buffer system A and B. Primary desorption was achieved by removal of (A) potassium sulfate or(B) ammonium sulfate only from eluent buffer systems. Secondary desorption was achieved by inclusion of 60% (v/v) ethylene glycol. Lane: (1) pool from desorption peak 1, buffer system A; (2) pool from desorption peak 2, buffer system A; (3) unbound proteins passed through with buffer system A; (4) pool from desorption peak 1, buffer system B; (5) pool from desorption peak 2, buffer system B; and (6) unbound proteins passed through with buffer system B. (Bottom-right) Electrophoresis on 1% agarose. Lanes l-6 correspond to the same samples at lanes l-6 on the 4/30 PAA gel (left). Lanes marked S show the pattern of whole unfractionated serum.

THIOPHILIC

ADSORPTION

OF IMMUNOGLOBULINS

225

It is important to mention here that separate preliminary investigations have demonstrated that the thiophilic column is not necessarily limited to an affinity for immunoglobulins. Table 2 shows a comparison of the relative thiophilic affinity of four other model proteins which are varied in size and surface charge (isoelectric points from 4.5 to 11). Conditions optimal for the thiophilic adsorption of individual proteins vary and further indicate the selectivity of the thiophilic adsorption process and potential for broader applications.

be easily modified by altering pH or concentration of water-structure-forming salt. Nearly quantitative recovery of sample and ease of column regeneration make routine use an efficient operation. The thiophilic adsorption mechanism is not known at this time. The results presented here indicate that in addition to the salt-promoted adsorption process, which has partially defined thiophilic gel character (5), a second adsorption event becomes prominent at especially low pH values or in the absence of waterstructure-forming salt. However, preliminary studies with other model proteins suggest that DISCUSSION salt-independent interaction with the T-gel at Affinity adsorption techniques are among neutral pH may be characteristic of only certhe most powerful procedures available for the tain proteins, including immunoglobulins, and large-scale isolation of biologically significant not a general phenomenon (11). In contrast, macromolecules. Furthermore, methodologies the increased adsorption observed at pH 3 is for the selective immobilization of biologically noted with all other proteins examined to date active proteins (e.g., receptors) and enzymes and is therefore likely to be a separate adsorphave consistantly proven invaluable for spe- tion property of the T-gel itself (11). Investicific applications and their uses are increasing gations underway (in preparation) with chroat both the analytical and preparative scale. matographically isolated pepsin and papain We have shown here that thiophilic adsorp- digestion products suggest that the F(abh and tion can be most selective and efficient for the F(ab) portions of human y-globulins may reimmobilization and/or purification of imtain some degree of thiophilic behavior, but munoglobulins. The relatively sharp transition their interaction with the T-gel may differ both in the thiophilic adsorption isotherms suggests quantitatively and qualitatively from that of nearly an all or nothing adsorption phenomthe intact immunoglobulins. It is possible that enon under the conditions studied. The high conditions may be determined for the selective adsorption capacity of the thiophilic gel can immobilization of these protein fragments. In this regard, we would like to emphasize that the selectivity of the thiophilic gel for other TABLE 2 proteins appears to be differentially optimized suggesting a potentially much broader specTHIOPHILIC BEHAVIOR OF OTHER MODEL PROTEINS trum of application than has been presented Concentration of here. Protein

Source

Lentil lectin Trypsin Trypsin inhibitor Lysozyme

Lens culinaris Bovine pancreas Soybean Chicken egg white Human serum

Immunoglobulins

(NH&SO4 required for 50% adsorption (70) 8-9 (w/v) 10-11 12 13 3-4

ACKNOWLEDGMENTS The authors thank Dr. Hans Bennich and Dr. Makonnen Belew for their time, comments, and suggestions during these and related studies. We thank Mrs. Birgit Olin for performing the electrophoresis experiments outlined in Fig. 8. The work has been financially supported by The Swedish Natural Science Research Council, The Swedish Board for Technical Development, the Ema and Victor Hasselblad Foundation, and LKB-Produkter AB.

226

HUTCHENS

REFERENCES 1. Ax&n, R., Porath, J., and Emback, S. (1967) Nature (London) 214, 1302-l 304. 2. Porath, J., and Ax&n, R. (1976) in Methods in Enzymology (Mosbach, K., ed.), Vol. 44, pp. 19-65, Academic Press, New York. 3. Porath, J., and Kristiansen, T. (1975) in The Proteins (Neurath, H., and Hill, R. L., eds.), Vol. 1,3rd ed. pp. 95-178, Academic Press, New York. 4. Porath, J., and Belew, M. (1983) in Affinity Chromatography and Biological Recognition (Chaiken, I. M., Wilchek, M., and Parikh, I., eds.), Academic Press, New York.

AND

PORATH

5. Porath, J., Ma&no, F., and Belew, M. (1985) FEBS Lett. 185, 306-310. 6. Porath, J., Sundberg, L., Fomstedt, N., and Olsson, I. (1973) Nature (London) 245,465-466. 7. Tiselius, A. (1948) Ark. Kemi Mineral. Geol. 26B, No. 1. 8. Porath, J. (1986) J. Chromatogr. Biomed. Appl. 376, 331-341. 9. Scatchard, G. (I 949) Ann. N. Y. Acad. Sci. 51, 660672.

10. Porath, J., L&s, T., and Janson, J.-C. (1975) J. ChrcF matogr.

103,49-62.

Il. Hutchens, T. W., and Porath, J., submitted for publications.