The Interaction of Proteins With Hydroxyapatite

ANALYTICAL BIOCHEMISTRY 136,425-432 (1984)

The Interaction of Proteins with Hydroxyapatite

I. Role of Protein Charge and Structure’

MARINAJ. GORBUNOFF

Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254

Received July 22, 1983

The criteria for elution of proteins from hydroxyapatite columns were examined as a function of (1) protein isoelectric point (22 proteins with isoelectric points between 3.5 and 11 .O); (2) ionic nature of eluant (Na salts of PO,, F-, Cl-, SCN-, ClO;, and CaCl& and (3) structural differences between related proteins. It was found that proteins can be classified into three groups: (1) basic proteins, which elute at similar, moderate molarities of PQ, F-, Cl-, SCN-, and ClOi, and low (<0.003 M) Ca”; (2) acidic proteins which elute at about equal moderate molarities of PO, and F-, but do not elute with Ca’+ and usually not with Cl-; (3) neutral proteins, which elute with PO.,, F-, and Cl-, but show a strong anion specificity, and do not elute with Car+ or SCN-. Furthermore, individual specific polar groups are not in general crucial to binding or desorption, and variations in structure, other than major loosening, do not influence strongly the pattern of protein-hydroxyapatite interaction.

KEY WORDS: hydroxyapatite; chromatography; protein separation.

Hydroxyapatite (HA)’ columns were orig- inally introduced by Tiselius et al. (1) for protein chromatography. Although they have been widely and successfully used since, particularly for the separation of nucleic acids (2,3), the mechanism of their operation is little understood. The principles of ion-exchange chromatography cannot be applied to HA columns, since the relation between protein affinity and their electrochemical behavior is not very pronounced in the case of HA (1,4). It is known that low molecular weight sub- stances, such as amino acids, are poorly, or not at all bound to HA (1,4-7). Synthetic

’ This work was supported by National Institutes of Health Grant GM 14603. This is Publication No. 1462 of the Graduate Department of Biochemistry Brandeis University.

’ Abbreviations used: HA, hydroxyapatite; Ct, chymotrypsin; BSA, bovine serum albumin; TPCK, L-l-to- sylamido-2-phenyl chloromethyl ketone; PMS-, phenyl- methanesulfonyl-; DIP-, diisopropylphosphoryl-; DES, des Tyr 146; i.e.p., isoelectric point; BCA, bovine carbonic anhydrase.

polypeptides of various molecular weights bind to HA, provided that they have a considerable total charge (2,7,8). A charge is, however, not a sufficient condition. While the affinity of polypeptides for HA is not affected by the presence of urea, proteins are not retained or very poorly retained when chro- matographed in urea (8). For a given ionic strength, the adsorption-desorption process is dependent on pH (5) and for a given pH on ionic strength (1,7). Thus, an increase in pH makes an eluant more effective, while, for the same pH, retention of a protein on the column takes place at low eluant molarity, with desorption at higher molarity.

In attempts to explain the operation of hydroxyapatite chromatography a number of mechanisms have been proposed. These in- clude (a) protein affinity for the calcium ions on the column (1); (b) the interaction between polar side groups on the protein and positive sites in the crystal (6); and (c) interaction between ionized residues on the protein and positive (calcium) or negative (phosphate) sites

425 0003-2697184 $3.00 Copyright 0 1984 by Academic f’rm. Inc. All rights of reproduction in any form reserved.

426 MARINA J. CORBUNOFF

on the column, depending on the net charge of the protein (7). Bernardi et al. (7) have probed into the mechanism of HA chromatography of macromolecules by studying the elution behavior of the biopolymers as a function of their isoelectric point, variation in the pH of the eluant, and variations in the chemical nature of the eluting buffers. The work presented below is a continuation of these studies. Sixteen additional proteins, covering the isoelectric point range from 3.5 to 11 .O have been examined, and the chemical nature of eluants has been extended by adding F- and ClO; ions to the previously used PO,, Cl-, and Ca2+ ions. Furthermore, the role of protein fine structure was investigated by comparing the elution behavior of chymotrypsins, chymotrypsinogens, and two genetic variants of @-lactoglobulin. The role of specific polar groups was probed by examining the elution behavior of a series of point modification products of a-chymotrypsin.

MATERIALS AND METHODS

Materials. Ovomucoid (Lot 335800), bovine carbonic anhydrase (Lot Ca8BA), lima bean trypsin inhibitor (OISFA), soybean trypsin inhibitor (ST1 5493), pepsinogen, crystalline (Lot PG6GA), trypsinogen (Lot TG8IE), chymotrypsinogen three times crystallized (Lot CDI36J835), a-chymotrypsin three times crystallized (Lot CID 36577 I), A-chymotrypsin (Lot CDD602), y-chymotrypsin two times crystallized (Lot CDG 6204-5), DIP-a-chymotrypsin (Lot CDDFP9CA), horseradish peroxidase (Lot 2559 HPOD 37H887), ribonuclease bovine Type IIA (Lot 7B1290), and lysozyme (Lot LY9CA) were purchased from Worthington. Conalbumin Type I (Lot 46C- 8 125), cytochrome c, horse heart, Type III HH (Lot 48B-7520), catalase, bovine liver (Lot 107C-870), a-la&albumin, and /3-lactoglobulin three times crystallized, were from Sigma Chemical Company. Myoglobin (horse heart), two times crystallized (Lot R 2042), insulin, bovine pancreas, recrystallized (Lot M 2 179), and bovine serum albumin, crystallized (Lot T 1533) were from Mann. Ovalbumin, five

times crystallized (Lot 15 1960 10) and hemoglobin, horse, two times crystallized (Lot HR 2262) were from Pentex. P-Lactoglobulins A and B were proteins (three times crystallized) prepared from the milk of individual cows (3653 and 4097). Papaya lysozyme was prepared according to Smith et al. (9).

The following derivatives of a-chymotryp sin were prepared as previously described: destyrosine- 146-, methylhistidine-57-, Met- 192-sulfoxide-, TPCK-, Tosyl-, and PMS- ( 10). Methylhistidine-57-destyrosine- 146-(u-chymotrypsin was prepared by methylation of destyrosine- 146-a-chymotrypsin. Neochy- motrypsinogen was prepared by a modified procedure of Valenzuela and Bender (11). The destyrosine- 146 derivatives of y-chymotrypsin and neochymotrypsinogen were prepared by the same methods as described for the CY- analog.

Phosphate buffers were prepared by mixing appropriate solutions of mono- and disodium phosphate. pH Adjustments were done at 25°C on a Model 4D radiometer pH meter.

Chromatographic experiments. A 1 X 20- cm HA column was used throughout. It was washed on a pump with at least 60 ml of the 0.001 M, pH 6.8, PO4 equilibrating buffer at a speed of 30 ml/h. This was followed by 60 ml 0.001 M NaCl whenever the column was used in the NaCl mode. One milliliter of protein solution (N 10 mg protein) was loaded onto the column and washed with two l-ml aliquots of the first eluant to be used; 2 ml of the same solvent was then introduced as a head on the column, and the column was washed with at least 30 ml of 0.00 1 M washing buffer. Elution was performed with solvents of pH 6.8 unless stated otherwise. Gradients were made by mixing 100-g portions of the appropriate solutions. Except for CaC12 experiments, the first chamber contained 0.01 M P04, while the second chamber was always 0.5 M in respect to the eluting salt plus 0.01 M P04. In CaC12 experiments, the protein solution was applied to a phosphate column. This was followed by washing with 30 ml of 0.001 M NaCl. The elution was performed either in shallow steps or with 0.00 1 M NaCl-

PROTEIN-HYDROXYAPATITE INTERACTIONS, I 427

0.005 M CaClz gradients when the isoelectric point of the protein was above 7. With acidic proteins, step elution with 0.5, 1.0, 2.0, and 3.0 M CaClz was used. The same technique was also used with NaCl. All salt solutions, except those of CaC&, were made up with 0.01 M, pH 6.8, PO4 buffer.

Retention under standard conditions is defined as retention on a phosphate column, when the material is not eluted with 30 ml of 0.001 M phosphate. Marginal retention is defined as retention on a NaCl column, when the material is not eluted with 30 ml of 0.00 1 M NaCl. The latter procedure is used only for material not retained under standard conditions.

RESULTS In Table 1 are compiled the results of the

elution behavior of 22 proteins having iso-

electric points between 3.5 and 11 .O. Gradients of sodium P04, F-, Cl-, and ClO; were used to examine the role of eluting anions. As can be seen the majority of proteins elute in the phosphate buffer within the molar&y range of 0.11 to 0.19, irrespective of their isoelectric points. Of the six proteins which do not fall within this range, two (papaya lysozyme and cytochrome c) are basic, and four (ovomucoid, bovine serum albumin, ovalbumin, and /I- lactoglobulin) are acidic. &Iactoglobulin, papaya lysozyme and cytochrome c elute at a PO4 molarity higher than 0.2. Of the low eluting proteins, ovomucoid is in a class by itself, since it displays marginal retention requiring a NaCl-washed column. Increasing the pH of the phosphate eluant to 7.8 makes elution somewhat easier for any value of the isoelectric point, while its decrease requires a higher mo-

TABLE 1

ELUTION OF PROTEINS FKOM HYDROXYAPATITE

El&on molarity

Nap04

Protein i.e.p. pH 6.8 pH 1.8 NaF NaCl NaSCN NaC104 CaCl*

Lima bean inhibitor 3.5-5.0 0.12 0.12 >3.0 >3.0 Ovomucoid 3.8-4.5 0.001 >3.0 >3.0 Pepsinogen 3.9 0.13 0.11 0.15 >3.0 >3.0 Soybean trypsin inhibitor 4.5 0.13 0.11 0.13 >3.0 13.0 cu-Lactalbumin 4.5 0.13 0.14 1.8 >3.0 Ovalbumin 4.6 0.09 0.12 >3.0 >3.0 Bovine serum albumin 4.1 0.06 0.05 0.13 >3.0 >3.0 @Lactoglubulin A” 5.1-5.3 0.24 0.66 >3.0 >3.0 Carbonic anhydrase B 5.3 0.11 0.12 0.10 0.21 >3.0 Catalase (bovine liver) 5.4 0.12 0.13 r3.0 Insulin 5.5 0.11 0.15 >3.0 >3.0 Conalbumin 6.8 0.18 0.13 >3.0 >3.0 Myoglobin 7.0 0.18 0.13 0.14 0.60 >3.0 >3.0 Horseradish peroxidase 1.2 0.17 Hemoglobin (horse) 1.6 0.19 0.10 0.66 >3.0 >3.0 a-Chymottypsin 8.1-8.6 0.18 0.24 0.26 0.25 0.003 Trypsinogen 9.3 0.17 0.13 0.23 0.32 0.31 >3.0 Chymotrypsinogen 9.5 0.19 0.15 0.24 0.23 0.19 0.002 Ribonuclease 9.1 0.12 0.13 0.13 0.17 0.001 Papaya lysozyme 10.5 0.25 0.20 0.25 0.24 0.001 Cytochrome c 10.6 0.25 0.35 0.32 0.24 0.002 Lysozyme 10.5-l I.0 0.18 0.14 0.19 0.18 0.15 0.15 0.001

a Elution was 0.29 at pH 6.4, >0.32 at pH 5.9 when PO., was the eluant.

428 MARINA J. GORBUNOFF

larity for elution, as shown for /3-lactoglobulin. All proteins are eluted by the fluoride ion. The effective molarity range is in general 0. lo- 0.24. Except for fi-lactoglobulin, acidic proteins, as a group, elute at the lower limit of this range (0.12-o. 15). Basic proteins, on the other hand, show considerable variability: horse hemoglobin (0.10) and ribonuclease (0.13) lie at the lower limit, while cytochrome c is at the top (0.35). fi-Lactoglobulin stands out, requiring 0.62 M F- for its elution.

Elution with the chloride ion reveals a new feature: basic proteins are eluted at about the same molarity as with the F- ion, while the majority of acidic proteins cannot be eluted even with 3 M Cl- ion. Of the three exceptions to this behavior only carbonic anhydrase and catalase are significant. For these two proteins the nature of the eluting agent does not seem to play any role, while a-la&albumin only confirms the inefficiency of Cl- ion as an eluant for acidic proteins. Furthermore, proteins having isoelectric points between 7 and 8 (myoglobin and hemoglobin) show a four- to sixfold increase in eluting molarity on going from the F- to the Cl- ion. These two proteins fail to elute when Cl- is replaced by SCN-. The same phenomenon is not observed for lysozyme (i.e.p. -10.8). Elution with per- chlorate ion of proteins having an i.e.p. of 8.1

to 10.8 does not show any new features when compared to P04, F-, or Cl- ions. Only one acidic protein (BCA)2 was studied. It eluted within the range found for basic proteins.

Elution with Ca” is characterized by a drastic difference between the elution behavior of proteins as a function of their isoelectric point. All acidic proteins, as well as those with isoelectric points between 7 and 8, do not elute with Ca2’ ion even at 3 M salt. Basic proteins, to the contrary, elute at a Ca2+ molarity of 0.001 to 0.003. Trypsinogen is the sole ex- ception. It is not eluted by 3 M Ca2+ ion.

Table 2 illustrates the effect of structure differences upon the adsorption-desorption behavior. p-Lactoglobulins A and B are examples of a slight variation in primary structure. The two genetic variants differ in amino acid com- position at two points in the sequence of the molecule. The most significant of these is re- placement of an aspartate residue in variant A by a glycine in variant B. Since the lactoglobulins exist as stable dimers in the pH range of interest, this means that &lactoglobulin A has two more COOH groups per kinetic unit than variant B. The elution behavior of the lactoglobulins is not identical. Both PO4 and F- ions are somewhat more effective as eluants for the B than the A variant.

The chymotrypsinogen-chymotrypsin

TABLE 2

EFFECT OF STRUCTURAL DIFFERENCES ON ELLJTION FROM HYDROXYAPATITE

Elution molarity

Protein PO4 NaF NaCl NaSCN NaC104 CaC12 -

b-Lactoglobulin A 0.24 0.66 r3.0 >3.0 (Xactoglobulin B 0.19 0.59 >3.0 >3.0

Chymotrypsinogen Neochymotrypsinogen Desneo-chymotrypsinogen

0.19 0.19 0.20

0.24 0.23 0.22 0.23

0.19 0.002 0.22 0.002

0.001

a-Chymotrypsin 0.18 0.24 0.25 A-chymotrypsin 0.20 0.24 0.26 y-Chymotrypsin 0.2 1 0.24 0.30

Lysozyme 0.18 Lysozyme, denatured 0.07

0.19 0.17 0.07

0.25 0.003 0.25 0.003 0.29 0.003

0.15 0.15 0.001 0.08 0.08


family is taken as an example primarily of variations in tertiary structure.3 The chymotrypsinogen set represents some of the possible forms of the zymogen. All changes in structure occur in the region of Tyr 146. The conformational transformation of chymotrypsinogen into neo-chymotrypsinogen is accompanied by the breaking of the Tyr 146-Thr 147 bond ( 12), while desneo-chymotrypsinogen can be produced by the artificial removal of Tyr 146. Neo- and desneo- can be assumed to have the same conformation. As can be seen, the elution behavior of these three compounds is practically identical. The chymotrypsin group comprises three forms of the active enzyme. The combined conformational and chemical changes involved in the zymogen to enzyme transformation result in A being identical to y, but not (Y in tertiary structure, while CY and y are identical in primary structure but different from the A enzyme. As can be seen the elution behavior of the three enzymes does not show any significant variations. It nevertheless differs from that of the zymogen set, as seen for the elutions in the ClO; and Ca2+ media. Furthermore, the enzymes show a slight ion specificity, with PO4 being a better eluant than Cl- or F- ions.

The example of lysozyme illustrates the effect of a profound loss of structure, as changes in secondary and tertiary structures associated with denaturation have curtailed quite sig- nificantly the ability of lysozyme to bind to HA. Furthermore, denatured STI, chymotrypsinogen, and cY-chymotrypsin were found to be totally unable to bind to HA.

Table 3 illustrates the effect of modification of a specific group upon the absorption-desorption process. The derivatives are isomorphous crystallographically with a-chymotryp sin ( 13,14). They differ from it by having a modifying group attached at a specific point. Methyl and TPCK are attached to histidine- 57; Tosyl, DIP and PMS to serine- 195. Des

3 The tertiary structure differences in all cases are in- duced by disruption of the primary structure, either by a break in the sequence or by deletion.

signifies the absence of Tyr-146, while Met- 192 the transformation of the methionine into its sulfoxide. As can be seen, this restricted modification has no effect on the elution behavior, except when it involves displacement of COOH groups along the three-dimensional structure (two last compounds), as both Des derivatives show ion selectivity and are closer to y-chymotrypsin than to the cx enzyme in their overall elution behavior.

Comparison of the effectiveness of P04, F-, Cl-, SCN-, and ClO, anions as eluants, reveals that basic proteins, having an i.e.p. above 8, show in general no significant variation in their elution behavior as one proceeds along the Hoffmeister series (Tables 1-3).4 Trypsinogen shows a considerable ion selectivity where PO4 is the best and Cl- is the worst eluant. Cy- tochrome c shows ion selectivity, where the order of eluants from strongest to weakest is ClO; > PO4 > Cl- > F-. There is a slight ion selectivity in the case of the chymotrypsins and the two Des derivatives of cY-chymotrypsin. At an isoelectric point between 7 and 8 the effectiveness of the anions becomes F > Cl- $ SCN-. On the acidic side, in the majority of cases, the relation becomes PO4 = F- b Cl-. The striking exceptions to this behavior are shown by catalase and bovine carbonic anhydrase-B. P-Lactoglobulin, although con- forming to the pattern of elution of acidic proteins with respect to the F B Cl relation, is nevertheless atypical. It elutes not only at the maximal PO4 molarity even for a basic protein, but it also exceeds all the studied proteins in the F- ion elution molarity. Fur- thermore, it is the only acidic protein for which the PO4 = F- relation does not hold.

4 The twofold increase in the elution molarity of basic proteins on going from PO, to NaCl or KC1 gradients, which had been reported previously (7), was found to be simply a reflection of the use of much steeper gradients in the case of chloride salts in the earlier study (7). Thus, in the present study, an elution molarity of 0.246 was obtained for lysozyme with a 0- 1.5 M PO, gradient in place of 0.18 M in the O-O.5 M gradient, while the NaCl molarity went up to 0.245 when a O-I .O M NaCl gradient was used in place of the one with 0.5 M.


TABLE 3

Emcr OF POINT MODIFICATION ON ELUTION

FROM HYDROXYAPATITE

Elution molarity

Protein PO4 NaF NaCI NaCIO, C&I*

a-Chymotrypsin 0.18 0.24 0.25 0.25 0.003 Tosyl-a-Ct 0.19 DIP-a-Ci 0.20 0.23 0.24 0.2 I 0.002 PMSa-Ct 0.19 0.21 0.22 0.21 0.002 FWK-c&t 0.22 Methyl-a-t3 0.18 0.24 0.23 0.2 I 0.0024 Met-192~ru-Ct 0.19 Des-a-ct 0.21 0.26 0.21 0.30 0.003 Methyl-Dew&t 0.22 0.26 0.3 1 0.27 0.003

DISCUSSION

The results described above permit to define more precisely the role of tertiary structure in the adsorption-desorption process. As shown by the examples of soybean trypsin inhibitor, chymotrypsinogen, cY-chymotrypsin, and lysozyme, the ability of proteins to bind to HA is completely lost or greatly diminished once their tertiary structure is loosened or reduced to the state of a random coil. On the other hand, structural changes which have very profound thermodynamic and biological conse- quences, may be of little consequence to the mechanism of operation of HA chromatography. The chymotrypsinogen-chymotrypsin family (Table 2) is a good example of this. The biological activity which arises as a consequence of the zymogen - enzyme transformation is dictated by changes in tertiary structure alone, and not related to any sub- sequent losses of a few individual amino acids ( 12). Identity in tertiary structure is the reason for the isomorphism of the A and y forms (12). The cy form of the enzyme is derived from neochymotrypsinogen by the same route as the A form from chymotrypsinogen (12). Since the alterations in primary structure which lead from A to y have already taken place in the zymogen - neochymotrypsinogen transformation, the y enzyme is identical to (Y in terms of primary structure. Their crys-

tallographic properties (12,15), as well as di- merization behavior in solution (10) prove, however, their nonidentity. The conformational difference between the two forms of the enzyme must be due to the fact that the sequence of events in their formation is not equivalent with respect to accommodation of steric strain. As can be seen from Table 2, none of these profound structural changes are reflected in any significant changes in the elution behavior within the chymotrypsinogen- chymotrypsin family. The only noticeable differences are when ClO; and Ca2+ are the eluants. Considering that only denaturation was effective in changing the elution behavior of proteins, one must conclude that the structural requirements of HA chromatography are only qualitatively rigorous. The case of ovomucoid might serve to indicate certain limits. It is a glycoprotein in which carbohydrate ac- counts for about 30% of the molecular weight. It has a very loose, diffise structure (16,17). It was the only native protein which was not adsorbed on HA under standard conditions.

The two genetic variants of B-lactoglobulin represent a case of conformational identity with slight variation in primary structure. The significant difference lies in the amino acid sequence (18) of the A variant (GluAsn- AspGlu, 62-65) which has one extra ionizable group per monomer subunit (19). Comparison of this pair could, therefore, answer the ques- tion whether a single carboxylate can affect the adsorption-desorption process. It should not be overlooked that the carboxyl in ques- tion is flanked by two more carboxyl groups. Since at pH 6.8 fi-lactoglobulin exists as a dimer, the A/B difference must be counted as two COOH groups. The elution behavior of the two variants certainly suggests that they are different. While the differences in the eluting molarities of the F- and particularly PO, ions are not large, they are significant. Since Asp 64 in the A variant has been suggested to exist as part of a cluster of carboxyls (20) on the surface of the protein, the stable dimer would have two such clusters. /M.actoglobulin must be considered as a special case on the


basis of its elution behavior: its eluting molarities of both PO4 and F- ions are not only in excess of those usually found for acidic proteins but even high for basic ones. &Lacto- globulin has been found to behave anoma- lously also in some of its other properties (2 1). Considering that carboxylic groups are im- plicated in the mechanism of operation of HA columns (6,7) it is tempting to relate this un- usual behavior to the singularity of the primary structure of fl-lactoglobulin. It contains several vicinal COOH groups in its amino acid sequence in addition to the above-mentioned 62-65 segment. They are GluGlu, 44-45; AspAspGlu, 129-131; GluGlu, 157-158 (18).

The role of specific individual ionizable groups in the adsorption-desorption process was tested on the derivatives of a-chymotrypsin, shown in Table 3. They comprise two derivatives of histidine-57 (PTCK, Methyl), three of serine-195 (tosyl, DIP, PMS); one of methionine-192; one (Des) lacks Tyr 146 and one (methyl-Des) is a derivative of histidine- 57 which also lacks Tyr 146. These derivatives are isomorphous with a-chymotrypsin in the crystal state (13,14), but not identical to it in solution, as shown by the acid self-association properties ( 10). There are small differences in structure between the crystalline and solution states (13). As can be seen from Table 3 the elution behavior of these eight derivatives does not show any significant difference when compared to that of cY-chymotrypsin, except for the appearance of slight ion selectivity in the case of the two derivatives in which a terminal COOH group has been displaced by one residue. Therefore, one must conclude that modification of single individual ionizable groups has little effect on the adsorption-desorption process even when a carboxyl group is involved, except when it forms part of a cluster, where the HA-COOH interaction would be cooperative. Such situations may exist in the P-lactoglobulins and in trypsinogen which has such a cluster at its C-terminal.

The results of this study permit the following conclusions:

I. The elution behavior of proteins as a

function of their isoelectric point can be sub- divided into three groups:

1. Basic proteins having isoelectric points above 8 elute with P04, F-, Cl-, SCN-, and ClO; at moderate molarities. Ion specificity, whenever shown, is not considerable. The generally lower eluting molarity of phosphate is probably related to the ionization state of the anion, although there is no correspondence with ionic strength. Except for trypsinogen, they elute at 0.003 M or lower molarity of Ca*+ ions.

2. Proteins, having isoelectric points between 7 and 8 elute with P04, F-, and Cl- ions, but not SCN- or Ca*+. They show a strong anion specificity F- > PO, 4 Cl- % SCN-.

3. Acidic proteins do not elute with Ca*+ ion. They elute at about equal molarities of PO4 and F- ions. The majority of them do not elute with Cl- ion.

The inverse relation between the pH of the eluant and the eluting molarity, reported by Bemardi et al. (7) for the phosphate eluant has been confirmed. This again can be related to the ionization state of the buffer and suggests a difference in the eluting strengths of the mono- and dibasic phosphate ions.

II. Only native proteins bind to HA effec- tively. A poorly defined, diffuse native structure, however, renders binding difficult.

III. Individual specific polar groups are in general not crucial either to the binding or desorption processes.

ACKNOWLEDGMENT

The author thanks Dr. G. Bemardi for proposing this problem, for his great interest, suggestions, and encour- agement during the performance of these studies, and for making available the facilities of his laboratory for carrying out the first part of this work.

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Documents

The Interaction of Proteins With Hydroxyapatite