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The use of trinitrobenzenesulfonic acid in studies on the binding of fatty acid anions to bovine serum albumin
ARCHIVES
OF
BIOCHEMISTRY
$ND
146, 428-440 (1971)
BIOPHYSICS
The Use of Trinitrobenzenesulfonic Binding
of
Fatty
Acid
Serum LARS-OLOV
ANDERSSON, Institute
JOHNNY
of Biochemistry,
Received
March
Acid Anions
in Studies
on the
to Bovine
Albumin BRANDT,
University
AND
of Uppsala,
16, 1971; accepted
April
SOLVEIG
Uppsala,
JOHANSSON
Sweden
22, 1971
The kinetics of the reaction between trinitrobenzenesulfonic acid (TNBS) and bovine serum albumin (BSA) have been studied. The results obtained indicate that TNBS is bound to certain binding sites on BSA and reacts with amino groups present there. The presence of palmitate strongly affects the rate of the reaction between TNBS and BSA probably by competition for the same binding sites. Tryptic digestion of BSA labeled with TNBS in the presence of various amounts of palmitate followed by fractionation of the peptides formed showed that labeling of one of the peptides was strongly inhibit)ed by the presence of palmitate. This peptide was isolated and further digested with chymotrypsin yielding a labeled pentapeptide. The sequence was determined and showed a tyrosine residue next to the labeled lysine. The binding affinity of caprylate and palmitate to native and TNBS-treated BSA was studied by equilibrium dialysis. A model for the binding of fatty acid anions to BSA is suggested:
The unusually strong affinity of serum albumin for anions and other substances has made it a model for studies on binding of various lowmolecular-weight substances. A large number of papers have been devoted to to this subject, land the binding constants and number of binding sites have been determined for many different substances. In spite of this there is not very much known about the actual mechanisms of binding and the nature of the binding sites. Studies on binding of fatty acids and various dyes indicate that electrostatic and hydrophobic interactions are involved, but detailed information about this is still not available. Some years ago Goldfarb (1,2) found that the amino groups of human serum albumin showed large variations in their reactivity towards trinitrobenzenesulfonic acid (TNBS) . From studies on the reaction kinetics using a large excess of TNBS he concluded that there were 1.5 amino groups which reacted very fast, 3.7 which reacted fast, and 14 which reactfed slow. The remain-
ing 39 amino groups of the 58 present in human serum albumin did not seem to react at all under the conditions used. To explain the large differences in reactivities Goldfarb suggested that binding of TNBS to serum albumin might occur before the reaction. Steric factors were also assumed to influence the reaction. In this study, the reactivity of some of the amino groups in bovine serum albumin (BSA) has been taken as a basis for a detailed study on the relationship between the binding of fatty acids to BSA and the reactivity of the amino groups. MATERIALS AND METHODS The bovine serum albumin sample used was obtained from Statens Bakteriologiska Laboratorium (SBL), Stockholm. It contained about 7-8% of dimer and polymer, and pure monomer was prepared by gel filtration on Sephadex G-150. The fatty acid content of the monomer, ss measured according to Dole (3), was 0.73 f 0.05 mole of fatty acid/mole of BSA. The molecular weight of monomeric serum albumin was assumed to be 66,000. 428
BINDING
OF FATTY
ACID
Defatted serum albumin was prepared according to Chen (4). The ratio charcoal (Norit) :BSA was 1:l. After defatting the pH of the solution was adjusted to about 5.0 with 0.2 M NaOH. Reduced and carboxymethylated BSA was prepared by reducing native BSA in 8 M urea at pH 9.0 for solution with 0.3 M mercaptoethanol 1 hr. Carboxymethylation was then accomplished by adding iocloacetate in slight excess of the amount of mercaptoethanol. The solution was allowed to stand in the dark for 24 hr at pH 8.5, and the low-molecular-weight substances were then removed by dialysis. Trypsin @CC-treated), chymotrypsin, and pepsin were obtained from Sigma Chem. Co. 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was also obtained from Sigma Chem. Co. It was dried in a desiccator before use. The trinitrophenpl derivative of glycine was prepared by allowing TNBS to react with an excess of glycine at pH 8.6 for 24 hr at room temperature. The ‘IC-labeled fatty acids, palmitic acid, and sodium caprylate were from Amersham. Studies on reaction kinetics. The reaction between TNBS and BSA was followed by measuring the increase in absorption at 350 nm which occurs when TNBS reacts with amino groups. To a 0.1% BSA solution in 0.05 M borate buffer in a cuvette was added the proper amount of 2 mM TNBS solution. The mixture was stirred and the cuvette was put in the spectrophotometer. Readings were made at intervals. The temperature was kept at 25 f 0.2”. Binding stufdiee. The equilibrium dialysis experiments were performed in 0.02 M phosphate buffer of pH 8.0. The concentration of protein in the dialysis bags was 2 mg/ml. The amount of solution in the bags was 3 ml, and it was allowed to equilibrate against 50ml of outer solution. The solutions were shaken for 48 hr at 5” to attain complete equilibrium. The dialysis bags were boiled and carefully washed before use. In the studies on picric acid and trinitrophenylated glycine (TNPglycine) the concentrations were determined by measuring the absorptions at 350 nm. The extinction coefficients of picric acid and TNP-glycine bound to BSA were determined by extrapolation of absorption measurements on a series of serum albumin solutions of various concentrations having the same concentration of picric acid or TNPglycine. The binding data obtained were plotted as I against F/C’~ -where c is the average number of bound molecules per protein molecule and C, is the corresponding equilibrium concentration of the substance bound. The number of binding sites and the association constants are obtained from the intercepts (5, 6). The association constants
TO SERUM
ALBUMIN
429
obtained are apparent constants K,, not corrected for electrostatic interactions and other factors. Binding of fatty acids was studied with Wlabeled sodium caprylate and palmitic acid. The concentrations of these were determined by applying 0.05 ml of solrLtion to a planchette and adding triethylamine to obtain an even film on drying. The solution was allowed to evaporate and the planchette was counted in a gas flow counter (Nuclear-Chicago). The concentration of fatty acid was then obtained by comparison with a calibration curve. The equilibrium dialysis was performed essentially as described above. Both inner and outer solutions were counted. In the studies on palmitate the procedure used B-as to dissolve the palmitate in the outer buffer solution at 5@60”, let it cool to about 35-40”, and then add the dialysis bag containing the albumin solution. Some hours later when the albumin had bound part of the palmitate the temperature was changed to 5”, and the solutions were shaken to obtain complete equilibrium at this temperature. The phosphate buffer used cont,ained trace amounts of sodium aside to prevent bacterial growth. A special procedure was used to study the binding of TNBS to BSA. To 50 ml of 0.2% albumin solution in 0.02 M phosphate buffer, pH 7.2, the proper amount of 5 rnM TNBS solution was added. The solution was stirred and subjected to ultrafiltration through a dialysis tubing which had been equilibrated with TNBS solution of the corresponding concentration. The first five 2-ml fractions of filtrate were collected, and the concentrations of TNBS were determined by adding an excess of glycine followed by absorption measurements at 350 nm after complete reaction. All solutions were kept at 5” except in the concentration determination step. The values obtained in the various fractions from the same run were about the same which indicates that the binding equilibrium is established rapidly, being essentially complete after 1 or 2 min. A complete binding curve was obtained by performing ultrafiltration experiments at a series of various TNBS concentrations. Amino acid analyses. The analyses were performed with a Spinco Model 120 D amino acid analyzer. The samples were hydrolyzed in sealed and evacuated Pyrex tubes for 24 hr with 6 M HCl at 110”. Chromatographic procedures. Sephadex G-50 gel filtrations were performed on 3.2 X 90-cm columns. The buffer used was 0.1 M ammonium acetate of pH 4.7. SE-Sephadex chromatography was performed on a 1.6 X 40-cm column. The gel was washed with 0.1 M NaOH and 0.1 M HCl prior to equilibration with 0.1 M acetic acid, 0.1 M formic acid buffer
430
ANDERSSON, BRANDT, AND JOHANSSON
adjusted to pH 3.0 with ammonia. The pHgradient elution was accomplished by connecting the column to a 700-ml reservoir of the 0.1 M acetic acid, 0.1 M formic acid buffer to which 0.2 M H,N solution was added continuously. The DEAE ion exchange chromatography was performed on Whatman DE-52 (Lot 2452740). The material was washed according to t,he manufacturer’s recommendations and equilibrated with 0.2 M phosphatebufferpH 7.5. A 1.6 X 20-cmcolumn wa8 packed and washed with 0.01 M phosphate buffer pH 7.5 before application of the sample. The elution was accomplished with a linear gradient 0.014.4 M phosphate buffer.
*A,,o 1
/
1.5
1.0
I/
/
I tJ 30
60 Time (min)
RESULTS
Reaction kinetics. The kinetics of the reaction between TNBS and BSA has been measured at different concentrations of TNBS. Figure 1 shows some of the curves obtained, plotted as log [(AAt,, - AA)/ AAt,J against time, where AAtOt is the total change in absorption at 350 nm taking place during the reaction and AA is the change in absorption between t = 0 and t = t. It is evident that the curves obtained are different. At the lowest concentration of TNBS the reaction is close to first order except for some deviation in the later part of the curve. On increasing the concentration of TNBS to 4 and 6 equiv per serum albumin the deviation from first order becomes more apparent. tliz for the reaction at two equivalents of TNBS per serum albumin is about 5.5 min Minutes
FIG. 1. Kinetics for the reaction between TNBS and BSA in 0.05 M borate buffer pH 8.4 at 25” plotted as log[(AAt,t - AA)/AAt,t] against time of reaction. AAtot is the total change in absorption at 350 nm taking place during the reaction and AA is the change in absorption between t = 0 and t = t. The concentration of BSA was 1.5 X 1C6 M and the concentrations of TNBS were: (0) 3.0 x lo-’ M, (X) 6.0 X lo- M, (0) 9.0 X lo- M,
FIG. 2. Kinetics (3.8 (0) 1&e The
for the reaction between X 10-S M) and (A) native BSA (7.6 X reduced and carboxymethylated BSA M). The buffer used was 0.05 M borate temperature was 25”.
TNBS 1O-6 M), (7.6 X pH 7.9.
corresponding to a first-order rate constant of 0.13 min-‘. Defatting of the BSA monomer by charcoal treatment yielded a sample that reacted faster with TNBS than the untreated sample. The reaction kinetics at 2 equiv of TNBS per mole of serum albumin were similar to that obtained with the nondefatted sample, but the rate was about 30 7%higher. The reaction between TNBS and BSA was strongly dependent on the conformation of the BSA. Reduced and carboxymethylated BSA showed a quite different curve than native BSA. Figure 2 shows the experimental curves for the reaction of native and reduced carboxymethylated BSA with TNBS. The concentration of TNBS is much higher here than in the previous experiments. The ratio c T~~s/C~~A is 500. Native BSA reacts more rapidly than the reduced carboxymethylated BSA in the earlier stages of t,he reaction but more slowly in the later stages. In his study on the reaction between TNBS and human serum albumin (HSA) Goldfarb (1, 2) suggested that binding of TNBS to the protein might occur before the reaction with the most reactive sites. If this is the case one could expect that other substances which are bound to BSA mi&t compete with the binding of TNBS and thereby influence the rate of the reaction. Experiments with palmitate present in the
BINDING
OF FATTY ACID TO SERUM ALBUMIN
*A350 0.2
20 10 Time (min) FIG. 3. Kinetics (3.0 X l(r5 M)
for the reaction
between
TNBS
and BSA (1.5 X 1O-6M) in the
presence of various fatty acid anions. The buffer used was 0.05 IK borate pH 8.2. Temperature 25”. (---) Native BSA without additions, (A) 3.0 X 10-4 M caproate, (0) 3.0 X W4 M caprylate, (0) 3.0 X KY4 M crtprinate, (X) 3.0 X 1O-5M laurate, (0) 3.0 X 10-S M palmitate. reaction mixture containing 2 equiv of TNBS per BSA were performed and showed that competition did occur. The rate of the reaction decreased considerably. This is compatible with the observation that defatted BSA reacts more rapidly with TNBS than native nondefatted 1BSA. It was then found, rather surprisingly, that the presence of caprylate had t,he opposite effect, i.e., the rate of the reaction was increased. This tempted a more systematic study of the influence of the size of the fatty acid. In Fig. 3 is shown the experimental curves obtained with various fatty acids present in the reaction mixtures of TNBS and BSA. It seems that the smaller fatty acids increase the rate of the reaction, the largest effect being obtained with caprylate (C,). This effect diminishes with increasing chain length and is nonexistent with laurate (C,,) which causes a small decrease of the rat,e. Further studies on the effects of caprylate on the reaction rate showed that as the concentration of TNBS is increased to 6.0 X 1O-5 :\I, corresponding to 4 equiv of TNBS per BSA, the effect of the caprylate disappeared. On further increase in the concentration of TNBS to 9.0 X low5 M the effect of the caprylate became the opposite, i.e., it decrea.sed the reaction rate. Labeling and characterization of reactive sites. To study which amino groups react with TNBS, experiments were performed
431
where BSA samples treated with various amounts of TNBS were hydrolyzed with trypsin followed by fractionation of the peptides formed. To 3.0 % BSA solutions in 0.2 M carbonate buffer pH 8.0 were added various amounts of 5 mM TNBS solution. The solutions were allowed to stand at room temperature overnight. Digestion was started by adding 1 mg of trypsin/lOO mg of BSA. Further additions of the same amount of trypsin were performed twice, 2 and 4 hr later. The pH of the solution was between 7.6 and 7.8. To stop disulfide exchange reactions and bacterial growth 0.5 ml of 0.01 M N-ethylmaleimid solution had been added. The hydrolysis was allowed to proceed for 12-15 hr. The pH of the solution was then adjusted to about 4.7 and the solution was applied to a Sephadex G-50 column. In Fig. 4 is shown the elution diagram obtained with a tryptic digest of a sample of native BSA which had been allowed to react with an equivalent amount of TNBS. The trinitrophenylated peptides are det’ected by their absorption at 350 nm. The diagram shows that there are four fractions containing most of the label. We have chosen to call them I-IV as indicated in the figure. Estimation of the molecular weights of the peptides in the various fractions from their elution volumes by comparison with a calibration
Effluent
volume
1
FIG. 4. Sephadex G-50 gel filtration elution diagram of a tryptic digest of 300 mg of native BSA that had been treated with an equivalent amount of TNBS.
432
ANDERSSON, ‘435, 0.2
BRANDT,
1 ctmitate Ktmitate
0.1 0 mitatexlmitate 0.1 0 0.1
ez Effluent
volume
1
FIG. 5. Sephadex G-50 gel filtration elution diagrams of tryptic digests of BSA samples that had been treated with TNBS under various conditions. The ratio CTNBS/CBSA = 1. The upper curve shows the labeling pattern obtained with native BSA + 3.0 equiv of palmitate. The curve in the middle is the pattern obtained with native BSA + 1.0 equiv of palmitate. The lower curve is the pattern obtained with defatted BSA.
curve plotted as log M, against the elution volume showed the following values: Fraction I 12,500, Fraction II 8,500, and the Fractions III and IV less than 2,000. The probeins used to obtain the calibration curve were cytochrome c, ribonuclease, myoglobin, and chymotrypsin. The elution diagrams obtained with the tryptic digests of the BSA samples that had been allowed to react with 2 and 3 equiv of TNBS were similar to the first one with the exception that the degree of trinitrophenylation had increased and that the relation between Fractions I and II was somewhat altered. Fraction II showed a larger increase than did Fraction I. The influence of various amounts of palmitate on the labeling pattern was studied. Figure 5 shows the elution diagrams obtained with tryptic digests of samples of native BSA that have been trinitrophenylated with TNBS in solutions to which have been added 1 and 3 equiv of palm&ate per BSA, respectively. The labeling pattern obtained with defatted BSA is also shown. It is evident that the presence of palmitate causes a decrease in the labeling of Fraction I, probably by competition with TNBS for a binding site on the protein. The other frac-
AND
JOHANSSON
tions do not seem to be affected. The decrease in labeling is most pronounced when 3 equiv of palmitate has been added. This might] indicate t-hat the two strongest binding sites (7) for palmitate are not involved in the reaction with TSBS. Further fractionatjion of bhe various labeled fractions was performed using SESephadex and DEAE-cellulose chromatography. The purity of the final peptide preparations were tested by polyacrylamide gel electrophoresis (8). In Fig. 6 is shown the elution diagram obtained upon SE-Sephadex fractionation of Fract,ion I from gel filtrat,ion of a tryptic digest of native BSA labeled with an equivalent amount of TKBS. One main and one minor TNBS-labeled pept,ide is seen, here called I, and Ib . Peptide I, was obtained essentially pure as indicated by further fractionation on DEAE-cellulose and purity tests by polyacrylamide gel electrophoresis. The amino acid composition is given in Table I. From the estimated molecular weight of the pept’ide it is possible to give the estimated number of residues. The peptide obtained is fairly large, mol wt 12,500, and consequently the amino acid composition does not give very much indication of the nature of the labeled site. Therefore experiments were performed in order to break down the peptide further. Complete reduction with 0.1 nr mercaptoethanol in borate buffer pH 9.0 containing 1.0 % sodium lauryl sulfate followed by carboxymethylat’ion with iodoacetic acid and gel filtration on
Tube number
FIG. 6. SE-Sephadex chromatography elation diagram of Fraction I from gel filtration of tryptic digest of native BSA that had been treated with an equivalent amount of TNBS. Elution patterns: (-) absorbance at 254 nm, (-O-O-) absorbance at 350 nm.
BINDING
OF FATTY
ACID TABLE
AMINO ACID
COMPOSITIONS
Amino acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine % Cystine Valine Methionine Isoleucine Leucine Tyrosine
Phenylalanine CM-cysteine Total
TO SERUM I OF VARIOUS
I&
CI
11.3 2.1 -
10.0 0.8 -
0.7 -
0.7 -
18.0 5.9 2.0 15.6 9.0 0.1 14.1 3.4 4.0 -
17.0 4.3 1.0
0.1 -
0.1 -
13.2 6.8 -
1.0 -
12.3 -
1.0 -
3.0 2.1
-
6.5 3.7 3.0 4.3
1.0 0.9 -
106
88
5
0.10 A:“58 0.15
FIG. 7. Sephadex G-25 gel filtration elution diagram of chymotryptic digest of peptide I, Elution patterns: (-O-O-) absorbance at 280 nm, (--t-W) absorbance at 350 nm.
Sephadex G-50 yielded a labeled peptide that contained 18 amino acid residues less than the original peptide as shown in Table I. Chymotryptic digestion of the pure peptide I, was then tried. About 4 mg of peptide was digested with 0.5 mg of chymotrypsin for 2 hr at pH 7.8 and room temperature. Figure 7 shows the Sephadex G-25 gel filtration elution diagram of the digest. The double peak appearing last in the diagram was subjected to SE-Sephadex chromatography and resolved into two components called C I and C II. The results of the amino acid analyses,
PEPTIDES
Residues/mole of peptide c II II,
Red. CM I,
1.9 6.7 5.7 4.0
433
ALBUMIN
1.0 0.1 1.0 0.9 4
IL
III,
10.5 3.4 1.1 15.5 5.8 3.9 16.7 6.0 2.9 9.3 7.2 3.6 1.0 1.0 7.9 3.7 4.2 -
8.6 1.8 1.3 12.1 5.2 2.8 12.4 6.2 2.0 6.0 5.2 2.5 1.0 1.1 7.2 1.8 4.1
107
82
7.9 1.5 0.2 8.0 2.2 4.2 9.7 3.6 1.0 2.6 5.3 2.0 3.6 6.0 1.6 1.1 63
which are given in Table I, clearly show that the two fractions are derived from the same part of peptide I, . They contain the same amino acids but C II contains one leucine residue less. N-t’erminal analysis using dansylation at, pH 8.5 followed by acid hydrolysis of the dansylated peptide and identification of the dansyl amino acids by thinlayer chromatography on polyamide sheet’s (9) showed that leucine was the N-terminal amino acid in the pentapeptide and alanine the N-terminal in the smaller peptide. Peptic digestion of peptide I, yielded a TK’BSlabeled tripeptide containing glutamic acid, alanine, and lysine. Alanine was the N-terminal amino acid. By digestion of this peptide for 5 days with carboxypeptidase A at pH 8.5 and at 5” followed by gel filtration on Sephadex G-25 it was possible to isolate a peptide having the same Rf value on paper chromatography as the dipept,ide Ala-Glu. These data only leave one possible sequence for the pentapeptide, the one shown below. TNP leu-ala-glu-lys-tyr
Peptide Ib was not obtained
completely
434
ANDERSSON.
BRANDT,
0.2 A 254 350
Tube number FIG. 8. SE-Sephadex chromatography elution diagram of Fraction II from gel filtration of tryptic digest of a native BSA sample that had been treated with an equivalent amount of TNBS. Elution pat,terns: (---) absorbance at 254 nm, (-a-@--) absorbance at 350 nm.
pure but the results of the amino acid analysis indicate that it is identical with the peptide II, described below. In Fig. 8 is shown the elution diagram of the SE-Sephadex fractionation of Fraction II from gel filtration of tryptic digest of trinitrophenylated BSA. It contains three major and two minor TNBS-labeled peptides. Further fractionation of the three main peptides by DEAE-cellulose chromatography yielded the pure peptides. Their amino acid compositions are shown in Table I. The data indicate that peptide IIt, is derived from peptide II, . A peptide containing 25 amino acids appears to be split off in the conversion of II, to Iii, . The peptides II, and 16, are not related to peptide I, . The molecular weights as calculated from the amino acid compositions are 12,100 for peptide II, and 9,200 for IIb . The value for II, is somewhat hight compared to the value 8,500 obtained from the gel filtration data. It has to be remembered, however, that molecular weight estimations by gel fiItration in ordinary buffer solutions can give erroneous values when the type of conformation of the protein or peptide is different from that of the proteins or peptides used for the calibration curve. In this case it seems clear that the molecular weights obtained from the amino acid analysis data are considerably more reliable than those obtained by gel filtration. The small size of the fragments, the purity, and the good amino acid analysis data ob-
AND
JOHANSSON
tained make Dhe estimation of number of residues fairly safe. Peptide II, is not related either to peptides II, and IL, or to peptide I, . It contains three disulfide bonds and has a molecular weight of 7200. It probably elutes on the rear side of the Fraction II peak. This peak is rather broad. SE-Sephadex chromatography of Fraction III resolved it int.0 five of six minor peaks. It was therefore decided not to study this fraction further, especially as it contains a rather small part of the total TNBS label. SE-Sephadex chromatography of Fraction IV yielded one main TNBS-labeled peptide and traces of two others. Further fractionation on DEAE-cellulose revealed another minor TNBS-IabeIed peptide and also separated t,he main part of a tyrosine-containing peptide from the main TNBS-peptide of this fraction. Amino acid analysis indicated the composition of the TNBS-peptide to be one asparagine, one lysine, and two alanine residues. Binding studies. The question of whether TNBS is bound to BSA before it reacts is somewhat difficult to test directly because the reaction interferes with the binding measurements. This was accomplished, however, by making use of special conditions as described in the hilaterials and Methods section. Ultrafiltration of various TNBS-BSA mixtures through dialysis tubing at 5” and p-5 e
I,
3
2
1
L
1
2
3
bJ5
FIG. 9. Scatchard plots of binding data obtained by ultrafiltration experiments where the binding of TNBS to native and trinitrophenylated BSA was studied at 5” in 0.02 M phosphate buffer pH 7.2. Native BSA (O), trinitrophenylated BSA containing 3.0 TNP groups/BSA (A).
BINDING
OF FATTY
ACID
pH 7.2, where the reaction between TNBS and BSA is slow, followed by measurements of the TNBS concentrations in the earlier fractions of the filtrate, yielded values from which the binding curve shown in Fig. 9 could be calculated. Under these conditions TNBS and BSA react only to a very limited extent during the lo-15 min employed for the ultrafiltration. The binding curve obtained indicates that there are two strong (K = 1.7 X lo5 &x-l) and three weak (K = 1.5 X lo4 M-~) binding sites. Some measurements were also performed at pH 6.S and 7.5. The values obtained were about the same as those obtained at pH 7.2. Experiments were performed in which the binding of TNBS to :BSA samples that had been reacted with various amounts of TNBS was studied. It was found that the Drinitrophenylation altered the binding properties of BSA considera,bly but that one strong binding site seemed.to be unaffected. Figure 9 shows the binding curve obtained with a BSA sa,mple which had been reacted with 3 equiv of TNBS per BSA. Further experiments with BSA reacted with various amounts of TNBS showed that most binding sites were affected except one. This indicates that there is one strong binding site for TNBS which does not contain any reactive amino group. In order to obtain information on why TNBS is bound to BSA, binding studies were made on some substances related to TNBS. The substances studied were picric acid, TNP-glycine, and trinitrobenzene. The technique used to determine the binding to BSA was the ordinary equilibrium dialysis. The binding data are given in Table II. Studies at pH 7.0 and 5” with picric acid showed about the same binding curve as at pH 8.0. These binding studies were limited to the concentration range of 5 X 1O-7-1O-4 M thereby neglecting low affinity binding. Some experiments were performed in which the effect of caprylate on the binding of TKP-glycine to native and trinitrophenylated BSA was studied. Caprylate competed, in fact, with TNP-glycine both for the strong and weak binding sites in native BSA but the competitive effect was stronger with trinitrophenylated BSA. From
TO SERUM
435
ALBUMIN TABLE
II
BINDING OF VARIOUS TRINITROPHENYL DERIVATIVES TO BSA AS STUDIED BY EQUILIBRIUM DIaLYSIS IN 0.02 M PHOSPHATE BUFFER PB 8.0 AT 5” Substance
Trinitrobenzene Picric acid TNP-glycine TNBSa D Determined
_____ nl
2
Kl(K’)
5 x
n2
Kz(@)
103
2 1.2 x 106 2 6 X lo5 2 1 1.7 x 106
2 2 3
by ultrafiltration
at pH 7.2.
105 106 1.5 x 104
the competitive effect of caprylate on the binding of TNP-glycine the association constant of caprylate for binding to the strong site that remains unaffected by the trinitrophenylation could be estimated to be about 2 X lo5 M-~. The binding of caprylate to other sites as obtained from the results shown below has been taken into account in the calculation. To study if the trinitrophenylation did affect the binding of fatty acids, experiments were performed in which the binding of caprylate and palmitate to native and trinitrophenylated BSA was compared. Equilibrium dialysis at 5” with 14C-labeled caprylate in 0.02 11 phosphate buffer pH 8.0 yielded binding curves which showed the presence of 1.6 moderately strong binding sites with K = 3.5 X lo5 M-I in native nondefatted BSA. Charcoal-defatted BSA showed 2.0 binding sites with K = 3.1 X lo5 b1-l. Trinitrophenylated BSA containing 3.0 trinitrophenyl groups per BSA shoxved the same number of strong binding sites and the same association constants as native BSA but less low--affinity binding of caprylate. Binding studies on palmitate and ot,her fatty acid anions of that size or bigger are usually performed in two-phase systems with one water phase and one hydrocarbon phase (7, 10). This is done in order to overcome the low solubility of these fatty acids in water and to be able to study a broad range of fatty acid concentrations. A disadvantage with this method is that the presence of the hydrocarbon solvent may introduce artifacts. There are indications of conformational
436
ANDERSSON,
BRANDT,
AND JOHANSSON
albumin obtained from AB Kabi, Stockholm, containing 0.92 equiv of bound fatty acids. The number of strong binding sites was 0.9 and the association constant was 3.1 X lo6 JI-’ which is only a slightly higher value than that obtained with BSA. The reason for the different results obtained in this study and the study by Goodman (7) is evidently to be found in the different methods used to determine the binding. Changes in conformation and/or motility. I To study whether changes in conformation 1 2 c and/or motility (13) occur on binding to or FIG. 10. Binding of palm&ate to native and modification of BSA the rate of enzymatic trinitrophenylated BSA as determined by equilibhydrolysis of BSA was studied. This is a rium dialysis in 0.02 M phosphate buffer pH 8.0 very sensitive method for detecting changes and 5”. Native BSA (O), trinitrophenylated BSA in conformation and motility (14, 15). The containing 3.0 TNP groups/BSA (A). hydrolytic enzymes used were trypsin and chymotrypsin. The rate of hydrolysis was changes of BSA caused by heptane dissolved followed by titration with 0.02 M NaOH soluin water (11). Another difficulty with the tion in an automatic titrator (Radiometer, method is to estimate the extent of dimerizaCopenhagen). The ratio mg enzyme/mg BSA tion of the fatty acid anions in the aqueous was l/100, the temperature 25”, and the pH phase (12). Because of the uncertainties S.O. The hydrolysis curves obtained were involved in the two-phase method, it was close to straight lines after 1 or 2 min after decided to use the ordinary equilibrium the initiation of the hydrolysis. The results dialysis method. This, however, limited the obtained are shown in Table III. It is clear study to high affinity binding. The binding that the pattern obtained is the same both curve obtained with native BSA at 5’ in 0.02 with trypsin and chymotrypsin. All subM phosphate buffer pH 8.0 is shown in Fig. stances studied do affect the rate of digestion 10. It indicates the presence of 1.3 strong binding sites with K = 2.2 X lo6 111-l.The but there seems to be no simple relation between the strength of the binding and the binding data obtained with trinitrophenyrate of digestion. The rate of hydrolysis of lated BSA containing 3.0 trinitrophenyl trinitrophenylated BSA is very low. The exgroups per BSA are about the same as with planation is probably that the covalent native BSA in the high affinity binding coupling of TNBS provides for a more effirange, but there seems to be a difference with cient stabilization. The fact that a few lysine respect to the further binding of palmitate. residues have been modified probably makes Unfortunately the limited concentration range attainable for the studies with the the rate of tryptic digestion lower, but this effect cannot be large as chymotryptic digespresent method does not permit a quantitation gives about the same results. tive evaluation of this further binding of palmitate. However, the data obtained DISCUSSION clearly show that the trinitrophenylation The kinetics of the reaction between does affect this type of binding. The association constant for the binding of TNBS and BSA at low concentrations of palmitate to the two strongest sites in BSA TNBS is close to first order. This is perhaps somewhat surprising because one might exobtained in this study is much lower than that for human serum albumin (K = 6 X pect a second-order rate behavior of this bimolecular reaction. If, however, TNBS is 10’ M-‘) as determined by the two-phase partition method (7). To clarify the reasons rapidly bound to BSA and reacts in the comfor this difference, some measurements were plexed form, a first-order behavior would be performed on a sample of human serum expected. That rapid binding of TNBS to
BINDING
OF FATTY
ACID
437
TO SERUM ALBUMIN
TABLE III BSA FGLUTIONS CONTAINING VARIOUS ADDED SUBSTANCESO
DIGESTION RATES OF NATIVE AND MODIFIED
Sample
Digestion rate with trypsin bmole/min) Digestion rate with chymotrypsin (I*mole/min)
BSA
TNP-BSA (3.0TNP/BSA)
~s$c$ gibp;ylate BSAcOT6 pd$de
BS.4 $.gpis
0.114
0.026
0.086
0.098
0.076
0.158
0.047
0.100
0.118
0.087
a Two per cent BSA solutions were digested with 1 to 100 of hydrolytic enzyme in 0.1 M N&l pH 8.0 and 25”. The digestion was followed by titration with 0.02 M NaOH in an automatic titrator.
BSA actually occurs is evident from the binding studies on the TNBS-BSA system. From these results and the observation that TNBS treatment blocks most of the binding sites for TNBS, it seems rather safe to conclude that at, low TNBS/BSA ratios the main part of the reaction occurs between TNBS bound to various binding sites and amino groups present in the vicinity of these sites. Reduction and carboxymethylation of BSA probably destroys the binding sites for TNBS and should accordingly lower the rat’e of the reaction with TNBS. This is in fact what is observed, viz., that although the total number of available amino groups is larger in reduced and carboxymethylated BSA, the initial rate of the reaction is slower than with native BSA. The studies on binding of substances similar to TNBS give some indications of the nature of the binding sites. First, it seems fairly obvious that picric acid, TNP-glycine, and trinitrobenzene are bound to binding sites that also1bind TNBS. The experimentally determined association constants show that the binding of trinitrobenzene is much weaker than the binding of the other substances. This is probably due to the fact that the other substances are ions and electrostatic interactions with ionic groups of the opposite charge on the protein contribute to the binding. Calculation of the free-energy change upon binding of trinitrobenzene to BSA from the apparent association constant yields the value 4.7 kcal/mole. This change in free energy results from the transfer of trinitr0benzen.e from water to binding sites in the protein. The binding of trinitrobenzene to BSA shou1.d probably be regarded as a
acid
at
pure hydrophobic bonding, and consequently it is possible to use the value 4.7 kcal/mole for the hydrophobic contribution in the binding of picric acid, TNBS, and TNPglycine. It is, however, possible that some hydrogen bonding may contribute to some extent to the binding of the trinitrobenzene to BSA. The electrostatic contribution to the freeenergy change upon binding of picric acid, TNBS, and TNP-glycine can be estimated by subtracting the value for the hydrophobic contribution from the total change in free energy on binding of these substances. This gives values in the range of 2.0-3.0 kcal/ mole. The value for picric acid is especially high. The conclusion that follows from these calculations and considerations is that hydrophobic and to a lesser degree electrostatic interactions are mainly responsible for the binding of picric acid, TNBS, and TNPglycine. This is very similar to the situation with binding of long-chain fatty acid anions (16) to serum albumin. The study has been limited to the pH range 7.0-8.4 thus being mainly concerned with binding at pH values close to that of the blood. In the studies on the kinetics of the reaction between TNBS and BSA it was found that the presence of fatty acids could affect the rate of the reaction in various ways depending on the size of the fatty acid added. Long-chain fatty acids such as palmitate decreased the rate of the reaction. The reason for this is probably that pahnitate is bound to one or several binding sites which may also bind and react with TNBS. The rapid reaction between TNBS and BSA at low
438
ANDERSSON,
BRANDT,
TNBS/BSA ratios depends on the binding of TNBS t’o binding sites containing amino groups, and it is quite clear that the rate of the reaction must diminish if a substance that is bound to one or several of these sites is added. This explanation is also in agreement with the observation made in connection with the labeling experiments that the addition of pahnitate caused a decrease in the degree of labeling of Fraction I. The increase of the reaction rate caused by caprylate is somewhat more difficult to explain. However, remembering the fact that there is one strong TNBS binding site that does not contain any reactive amino group it is possible to find a reasonable explanation. If this nonreactive TNBS binding site also binds caprylate, the addition of caprylate to the solution would increase the concentration of free TNBS because of the competitive binding. If caprylate is not at all, or weakly, bound to the reactive TNBS binding sites, this would result in an increase in the rate of the reaction between TNBS and BSA. Further support for this explanation is obtained from the fact that on increasing the TNBS/BSA ratio the rate-increasing effect of caprylate disappears and it becomes slightly rate decreasing. This indicates that caprylate is in fact weakly bound to the reactive TNBS binding sites. The results of the studies on competitive binding of TNPglycine and caprylate bo native and TNBStreated BSA are in agreement with the explanations suggested above. The binding of TNBS and related substances to serum albumin shows many similarities to the binding of fatty acid anions. The studies on the labeling of binding sites with TNBS shows that one of the most reactive sites is involved in the binding of palmitate. The observation that defatting increases the reactivity of this site might be taken as an indication that it is one of the two strongest binding sites for palmitate. However, the results from the studies on the binding of palm&ate to native and trinitrophenylated BSA show that the two strongest binding sites are not involved, but that probably one of the moderately strong binding sites is blocked by the trinitrophenylation. It thus appears as if the reactive site in the
AND JOHANSSON
Fraction I peptide is one of the moderately strong binding sites for long-chain fatty acids. The results of the labeling experimenbs with various amounts of palmitate present in the reaction mixture are also in agreement with this conclusion. The amino acid sequence around the reactive lysine shows some interesting features. The lysine residue has on one side a glutamic acid residue with which it probably interacts and on the other side a tyrosine residue. The presence of this tyrosine residue immediately adjacent to the lysine should probably be correlated with the observation (16) that the binding of fatty acids induced shifts in the uv-spectrum of BSA which are characteristic for changes in tyrosine environment. In connection with studies on the binding of 1-anilino-S-naphthalene sulfonic acid (ANS) to BSA, Anderson and Weber suggested (17) that ANS was bound to clefts between various globular parts of BSA. These globular parts have been called “subunits,” and there are indications from several types of measurements (E-20) that the peptide chain of serum albumin is folded so as to constitute three or four globular parts (20), the subunits. It is an attractive hypothesis that binding of fatty acid anions should occur to clefts between subunits. If the inner parts of the clefts are hydrophobic and some lysine or arginine residue is situated on the outer part of the cleft a very favorable situation for binding would be present. Hydrophobic and electrostatic bonding would be the main binding forces as is the case on binding of long-chain fatty acids (16) and detergents (21). The subunit hypothesis suggests that there is some degree of flexibility between the subunits. This can explain several different observed effects of fatty acids and detergents on serum albumin. The binding of a fatty acid anion to a cleft between two subunits would certainly diminish the freedom of movement of the two subunits versus each other. Another effect closely linked to this is the stabilization of the whole protein molecule obtained by not exposing the hydrophobic parts of the binding site to water. These two effects, the stabilization and the
BINDING
OF FATTY
ACID
decreased motility, are in agreement with the observations that long-chain fatty acids stabilize serum albumin against heat denaturation (22) and that binding of fatty acid anions and similar substances makes the albumin less susceptible to attack by proteolytic enzymes (23). Measurements of optical rotation have shown (24) that the binding of small amounts of detergents does affect the conformation of serum albumin. It is possible that binding to the clefts between the subunits is a general mechanism for the binding of various substances to serum albumin. The conformation and motility changes can also be put in relation to some observations made on the isoelectric focusing behavior of serum albumin, in which it was found that the binding of fatty acid anions greatly influenced the p1 of the protein (25). The p1 diminished from about 5.8 to 4.7 on binding of fatty acid anions. A possible explanation is that the conformational changes taking place upon binding, among other things, also buries some amino groups. Indications for the presence of buried amino and carboxyl groups in native BSA have been obtained by Avruch et al. (26) who found that acetimidation of the lysine residues in BSA altered the charge and the carboxyl titration behavior of BSA. Vijai and Foster (27) have suggested that pairs of unionized carboxyl and. amino groups are situated in the clefts between the subunits of BSA. The main features of the binding mechanism suggested are indicated below using a simplified section model of serum albumin with two subunits. Hydrophobic areas are shaded.
Defat.ted st,ate High motility Low stability
Fatty acid containing state Low motility High stability
TO SERUM ALBUMIN
439
One of the assumptions made in the derivation of the Scatchard equation (5, 6) for binding to multiple sites is that all sites behave completely independently of each other. This is probably not the case in the binding of fatty acids and detergents to serum albumin. Both the observed changes in conformation and motility and the nature of the subunit model strongly indicate that binding to one or several sites has an influence on the binding to the remaining sites. From this it follows that the values obtained for the number of binding sites and binding constants must be regarded as somewhat uncertain. The increase in TNBS labeling of Fraction I when defatted BSA is used may be due t’o general conformation or motility changes upon defatting which also affect sites not containing fatty acid originally. ACKNOWLEDGMENTS We thank Dr. C-G Rosen for valuable discussion and criticism of the manuscript. The study has been supported by grants from Swedish Board for Technical Development. REFERENCES 1. 2.
GOLDFARB, GOLDFARB,
A. R., Biochemistry 6, 2574 (1966). A. R., Biochim. Biophys. Acta 200,
1 (1970). 3. DOLE, V. P., J. Clin. Invest. 35, 150 (1956). 4. CHEN, R. F., J. Biol. Chem. 242, 173 (1967). G., SCHEINBORG, I. H., AND 5. SCATCHARD, ARMSTRONG, S. H., J. Amer. Chem. Sot. 72, 535, 540 (1950). 6. SCATCHARD, G., COLEMAN, J. B., AND SHEN, A. L., J. Amer. Chem. Sot. 79, 17 (1957). 7. GOODMAN,D. S., J. Amer. Chem. Sot. 60, 3892 (1958). 8. RAYMOND, S., Ann. N. Y. Acad. Sci. 121, 350 (1964). 9. WOODS, K. R., AND WANG, K.-T., Biochim. Biophys. Acta 133, 369 (1967). 10. ARVIDSSON, E. O., Small molecule-protein interactions, Thesis, Studentlitteratur, Lund (1965). Il. ALFS~N, A., C. R. Trav. Lab. Carlsberg 33, 415 (1963). 12. MUKERJEE, P., J. Phys. Chem. 69, 2821 (1965). 13. TANIUCHI, H., MORAVEK, L., AND ANFINSEN, C. B., J. Biol. Chem. 244, 4600 (1969). 14. MARKUS, G., Proc. Nat. Ad. Sci. U. S. A. 64, 253 (1965).
440
ANDERSSON,
BRANDT,
15. MARKUS, G., MCCLINTOCK, II. K., AND CASTELLINI, B. A., J. Biol. Chem. 242, 4402 (1967). 16. REYNOLDS, J., HERBERT, S., AND STEINHARDT, J., Biochemistry 7, 1357 (1968). 17. ANDERSON, S. R., AND WEBER, G., Biochemistry 8, 371 (1969). 18. BLOOMFIELD, V., Biochemistry 6, 684 (1966). 19. WEBER, G., AND YOUNG, L. B., J. Biol. Chews. 289, 1424 (1964). 20. PEDERSON, D. M., AND FOSTER, J. F., Biochemistry 8, 2357 (1969). 21. RAY, A., REYNOLDS, J. A., POLET, H., AND STEINHARDT, J., Biochemistry 6, 2606 (1966).
AND
JOHANSSON
22. BOYER, P. D., LUM, F. G., BALLOU, G. A., LUCK, J. M., AND RICE, R. G., J. Biol. Chem. 162, 181 (1946). 23. KLEPPER, J. A., JR., AND CANN, J. R., Arch. Biochem. Biophys. 108, 531 (1964). 24. REYNOLDS, J. A., HERBERT, S., POLET, H.! AND STEINHARDT, J., Biochemistry 6, 937 (1967). 25. VALMET, E., in “Protides of Biological Fluids” (H. Peeters, ed.), Vol. 17, p. 443 (1969). 26. AVRUCH, J., REYNOLDS, J. A., AND REYNOLDS J. H., Biochemistry 8, 1855 (1969). 27. VIJAI, K. K., AND FOSTER, J. F., Biochemistry 6, 1152 (1967).
OF
BIOCHEMISTRY
$ND
146, 428-440 (1971)
BIOPHYSICS
The Use of Trinitrobenzenesulfonic Binding
of
Fatty
Acid
Serum LARS-OLOV
ANDERSSON, Institute
JOHNNY
of Biochemistry,
Received
March
Acid Anions
in Studies
on the
to Bovine
Albumin BRANDT,
University
AND
of Uppsala,
16, 1971; accepted
April
SOLVEIG
Uppsala,
JOHANSSON
Sweden
22, 1971
The kinetics of the reaction between trinitrobenzenesulfonic acid (TNBS) and bovine serum albumin (BSA) have been studied. The results obtained indicate that TNBS is bound to certain binding sites on BSA and reacts with amino groups present there. The presence of palmitate strongly affects the rate of the reaction between TNBS and BSA probably by competition for the same binding sites. Tryptic digestion of BSA labeled with TNBS in the presence of various amounts of palmitate followed by fractionation of the peptides formed showed that labeling of one of the peptides was strongly inhibit)ed by the presence of palmitate. This peptide was isolated and further digested with chymotrypsin yielding a labeled pentapeptide. The sequence was determined and showed a tyrosine residue next to the labeled lysine. The binding affinity of caprylate and palmitate to native and TNBS-treated BSA was studied by equilibrium dialysis. A model for the binding of fatty acid anions to BSA is suggested:
The unusually strong affinity of serum albumin for anions and other substances has made it a model for studies on binding of various lowmolecular-weight substances. A large number of papers have been devoted to to this subject, land the binding constants and number of binding sites have been determined for many different substances. In spite of this there is not very much known about the actual mechanisms of binding and the nature of the binding sites. Studies on binding of fatty acids and various dyes indicate that electrostatic and hydrophobic interactions are involved, but detailed information about this is still not available. Some years ago Goldfarb (1,2) found that the amino groups of human serum albumin showed large variations in their reactivity towards trinitrobenzenesulfonic acid (TNBS) . From studies on the reaction kinetics using a large excess of TNBS he concluded that there were 1.5 amino groups which reacted very fast, 3.7 which reacted fast, and 14 which reactfed slow. The remain-
ing 39 amino groups of the 58 present in human serum albumin did not seem to react at all under the conditions used. To explain the large differences in reactivities Goldfarb suggested that binding of TNBS to serum albumin might occur before the reaction. Steric factors were also assumed to influence the reaction. In this study, the reactivity of some of the amino groups in bovine serum albumin (BSA) has been taken as a basis for a detailed study on the relationship between the binding of fatty acids to BSA and the reactivity of the amino groups. MATERIALS AND METHODS The bovine serum albumin sample used was obtained from Statens Bakteriologiska Laboratorium (SBL), Stockholm. It contained about 7-8% of dimer and polymer, and pure monomer was prepared by gel filtration on Sephadex G-150. The fatty acid content of the monomer, ss measured according to Dole (3), was 0.73 f 0.05 mole of fatty acid/mole of BSA. The molecular weight of monomeric serum albumin was assumed to be 66,000. 428
BINDING
OF FATTY
ACID
Defatted serum albumin was prepared according to Chen (4). The ratio charcoal (Norit) :BSA was 1:l. After defatting the pH of the solution was adjusted to about 5.0 with 0.2 M NaOH. Reduced and carboxymethylated BSA was prepared by reducing native BSA in 8 M urea at pH 9.0 for solution with 0.3 M mercaptoethanol 1 hr. Carboxymethylation was then accomplished by adding iocloacetate in slight excess of the amount of mercaptoethanol. The solution was allowed to stand in the dark for 24 hr at pH 8.5, and the low-molecular-weight substances were then removed by dialysis. Trypsin @CC-treated), chymotrypsin, and pepsin were obtained from Sigma Chem. Co. 2,4,6-Trinitrobenzenesulfonic acid (TNBS) was also obtained from Sigma Chem. Co. It was dried in a desiccator before use. The trinitrophenpl derivative of glycine was prepared by allowing TNBS to react with an excess of glycine at pH 8.6 for 24 hr at room temperature. The ‘IC-labeled fatty acids, palmitic acid, and sodium caprylate were from Amersham. Studies on reaction kinetics. The reaction between TNBS and BSA was followed by measuring the increase in absorption at 350 nm which occurs when TNBS reacts with amino groups. To a 0.1% BSA solution in 0.05 M borate buffer in a cuvette was added the proper amount of 2 mM TNBS solution. The mixture was stirred and the cuvette was put in the spectrophotometer. Readings were made at intervals. The temperature was kept at 25 f 0.2”. Binding stufdiee. The equilibrium dialysis experiments were performed in 0.02 M phosphate buffer of pH 8.0. The concentration of protein in the dialysis bags was 2 mg/ml. The amount of solution in the bags was 3 ml, and it was allowed to equilibrate against 50ml of outer solution. The solutions were shaken for 48 hr at 5” to attain complete equilibrium. The dialysis bags were boiled and carefully washed before use. In the studies on picric acid and trinitrophenylated glycine (TNPglycine) the concentrations were determined by measuring the absorptions at 350 nm. The extinction coefficients of picric acid and TNP-glycine bound to BSA were determined by extrapolation of absorption measurements on a series of serum albumin solutions of various concentrations having the same concentration of picric acid or TNPglycine. The binding data obtained were plotted as I against F/C’~ -where c is the average number of bound molecules per protein molecule and C, is the corresponding equilibrium concentration of the substance bound. The number of binding sites and the association constants are obtained from the intercepts (5, 6). The association constants
TO SERUM
ALBUMIN
429
obtained are apparent constants K,, not corrected for electrostatic interactions and other factors. Binding of fatty acids was studied with Wlabeled sodium caprylate and palmitic acid. The concentrations of these were determined by applying 0.05 ml of solrLtion to a planchette and adding triethylamine to obtain an even film on drying. The solution was allowed to evaporate and the planchette was counted in a gas flow counter (Nuclear-Chicago). The concentration of fatty acid was then obtained by comparison with a calibration curve. The equilibrium dialysis was performed essentially as described above. Both inner and outer solutions were counted. In the studies on palmitate the procedure used B-as to dissolve the palmitate in the outer buffer solution at 5@60”, let it cool to about 35-40”, and then add the dialysis bag containing the albumin solution. Some hours later when the albumin had bound part of the palmitate the temperature was changed to 5”, and the solutions were shaken to obtain complete equilibrium at this temperature. The phosphate buffer used cont,ained trace amounts of sodium aside to prevent bacterial growth. A special procedure was used to study the binding of TNBS to BSA. To 50 ml of 0.2% albumin solution in 0.02 M phosphate buffer, pH 7.2, the proper amount of 5 rnM TNBS solution was added. The solution was stirred and subjected to ultrafiltration through a dialysis tubing which had been equilibrated with TNBS solution of the corresponding concentration. The first five 2-ml fractions of filtrate were collected, and the concentrations of TNBS were determined by adding an excess of glycine followed by absorption measurements at 350 nm after complete reaction. All solutions were kept at 5” except in the concentration determination step. The values obtained in the various fractions from the same run were about the same which indicates that the binding equilibrium is established rapidly, being essentially complete after 1 or 2 min. A complete binding curve was obtained by performing ultrafiltration experiments at a series of various TNBS concentrations. Amino acid analyses. The analyses were performed with a Spinco Model 120 D amino acid analyzer. The samples were hydrolyzed in sealed and evacuated Pyrex tubes for 24 hr with 6 M HCl at 110”. Chromatographic procedures. Sephadex G-50 gel filtrations were performed on 3.2 X 90-cm columns. The buffer used was 0.1 M ammonium acetate of pH 4.7. SE-Sephadex chromatography was performed on a 1.6 X 40-cm column. The gel was washed with 0.1 M NaOH and 0.1 M HCl prior to equilibration with 0.1 M acetic acid, 0.1 M formic acid buffer
430
ANDERSSON, BRANDT, AND JOHANSSON
adjusted to pH 3.0 with ammonia. The pHgradient elution was accomplished by connecting the column to a 700-ml reservoir of the 0.1 M acetic acid, 0.1 M formic acid buffer to which 0.2 M H,N solution was added continuously. The DEAE ion exchange chromatography was performed on Whatman DE-52 (Lot 2452740). The material was washed according to t,he manufacturer’s recommendations and equilibrated with 0.2 M phosphatebufferpH 7.5. A 1.6 X 20-cmcolumn wa8 packed and washed with 0.01 M phosphate buffer pH 7.5 before application of the sample. The elution was accomplished with a linear gradient 0.014.4 M phosphate buffer.
*A,,o 1
/
1.5
1.0
I/
/
I tJ 30
60 Time (min)
RESULTS
Reaction kinetics. The kinetics of the reaction between TNBS and BSA has been measured at different concentrations of TNBS. Figure 1 shows some of the curves obtained, plotted as log [(AAt,, - AA)/ AAt,J against time, where AAtOt is the total change in absorption at 350 nm taking place during the reaction and AA is the change in absorption between t = 0 and t = t. It is evident that the curves obtained are different. At the lowest concentration of TNBS the reaction is close to first order except for some deviation in the later part of the curve. On increasing the concentration of TNBS to 4 and 6 equiv per serum albumin the deviation from first order becomes more apparent. tliz for the reaction at two equivalents of TNBS per serum albumin is about 5.5 min Minutes
FIG. 1. Kinetics for the reaction between TNBS and BSA in 0.05 M borate buffer pH 8.4 at 25” plotted as log[(AAt,t - AA)/AAt,t] against time of reaction. AAtot is the total change in absorption at 350 nm taking place during the reaction and AA is the change in absorption between t = 0 and t = t. The concentration of BSA was 1.5 X 1C6 M and the concentrations of TNBS were: (0) 3.0 x lo-’ M, (X) 6.0 X lo- M, (0) 9.0 X lo- M,
FIG. 2. Kinetics (3.8 (0) 1&e The
for the reaction between X 10-S M) and (A) native BSA (7.6 X reduced and carboxymethylated BSA M). The buffer used was 0.05 M borate temperature was 25”.
TNBS 1O-6 M), (7.6 X pH 7.9.
corresponding to a first-order rate constant of 0.13 min-‘. Defatting of the BSA monomer by charcoal treatment yielded a sample that reacted faster with TNBS than the untreated sample. The reaction kinetics at 2 equiv of TNBS per mole of serum albumin were similar to that obtained with the nondefatted sample, but the rate was about 30 7%higher. The reaction between TNBS and BSA was strongly dependent on the conformation of the BSA. Reduced and carboxymethylated BSA showed a quite different curve than native BSA. Figure 2 shows the experimental curves for the reaction of native and reduced carboxymethylated BSA with TNBS. The concentration of TNBS is much higher here than in the previous experiments. The ratio c T~~s/C~~A is 500. Native BSA reacts more rapidly than the reduced carboxymethylated BSA in the earlier stages of t,he reaction but more slowly in the later stages. In his study on the reaction between TNBS and human serum albumin (HSA) Goldfarb (1, 2) suggested that binding of TNBS to the protein might occur before the reaction with the most reactive sites. If this is the case one could expect that other substances which are bound to BSA mi&t compete with the binding of TNBS and thereby influence the rate of the reaction. Experiments with palmitate present in the
BINDING
OF FATTY ACID TO SERUM ALBUMIN
*A350 0.2
20 10 Time (min) FIG. 3. Kinetics (3.0 X l(r5 M)
for the reaction
between
TNBS
and BSA (1.5 X 1O-6M) in the
presence of various fatty acid anions. The buffer used was 0.05 IK borate pH 8.2. Temperature 25”. (---) Native BSA without additions, (A) 3.0 X 10-4 M caproate, (0) 3.0 X W4 M caprylate, (0) 3.0 X KY4 M crtprinate, (X) 3.0 X 1O-5M laurate, (0) 3.0 X 10-S M palmitate. reaction mixture containing 2 equiv of TNBS per BSA were performed and showed that competition did occur. The rate of the reaction decreased considerably. This is compatible with the observation that defatted BSA reacts more rapidly with TNBS than native nondefatted 1BSA. It was then found, rather surprisingly, that the presence of caprylate had t,he opposite effect, i.e., the rate of the reaction was increased. This tempted a more systematic study of the influence of the size of the fatty acid. In Fig. 3 is shown the experimental curves obtained with various fatty acids present in the reaction mixtures of TNBS and BSA. It seems that the smaller fatty acids increase the rate of the reaction, the largest effect being obtained with caprylate (C,). This effect diminishes with increasing chain length and is nonexistent with laurate (C,,) which causes a small decrease of the rat,e. Further studies on the effects of caprylate on the reaction rate showed that as the concentration of TNBS is increased to 6.0 X 1O-5 :\I, corresponding to 4 equiv of TNBS per BSA, the effect of the caprylate disappeared. On further increase in the concentration of TNBS to 9.0 X low5 M the effect of the caprylate became the opposite, i.e., it decrea.sed the reaction rate. Labeling and characterization of reactive sites. To study which amino groups react with TNBS, experiments were performed
431
where BSA samples treated with various amounts of TNBS were hydrolyzed with trypsin followed by fractionation of the peptides formed. To 3.0 % BSA solutions in 0.2 M carbonate buffer pH 8.0 were added various amounts of 5 mM TNBS solution. The solutions were allowed to stand at room temperature overnight. Digestion was started by adding 1 mg of trypsin/lOO mg of BSA. Further additions of the same amount of trypsin were performed twice, 2 and 4 hr later. The pH of the solution was between 7.6 and 7.8. To stop disulfide exchange reactions and bacterial growth 0.5 ml of 0.01 M N-ethylmaleimid solution had been added. The hydrolysis was allowed to proceed for 12-15 hr. The pH of the solution was then adjusted to about 4.7 and the solution was applied to a Sephadex G-50 column. In Fig. 4 is shown the elution diagram obtained with a tryptic digest of a sample of native BSA which had been allowed to react with an equivalent amount of TNBS. The trinitrophenylated peptides are det’ected by their absorption at 350 nm. The diagram shows that there are four fractions containing most of the label. We have chosen to call them I-IV as indicated in the figure. Estimation of the molecular weights of the peptides in the various fractions from their elution volumes by comparison with a calibration
Effluent
volume
1
FIG. 4. Sephadex G-50 gel filtration elution diagram of a tryptic digest of 300 mg of native BSA that had been treated with an equivalent amount of TNBS.
432
ANDERSSON, ‘435, 0.2
BRANDT,
1 ctmitate Ktmitate
0.1 0 mitatexlmitate 0.1 0 0.1
ez Effluent
volume
1
FIG. 5. Sephadex G-50 gel filtration elution diagrams of tryptic digests of BSA samples that had been treated with TNBS under various conditions. The ratio CTNBS/CBSA = 1. The upper curve shows the labeling pattern obtained with native BSA + 3.0 equiv of palmitate. The curve in the middle is the pattern obtained with native BSA + 1.0 equiv of palmitate. The lower curve is the pattern obtained with defatted BSA.
curve plotted as log M, against the elution volume showed the following values: Fraction I 12,500, Fraction II 8,500, and the Fractions III and IV less than 2,000. The probeins used to obtain the calibration curve were cytochrome c, ribonuclease, myoglobin, and chymotrypsin. The elution diagrams obtained with the tryptic digests of the BSA samples that had been allowed to react with 2 and 3 equiv of TNBS were similar to the first one with the exception that the degree of trinitrophenylation had increased and that the relation between Fractions I and II was somewhat altered. Fraction II showed a larger increase than did Fraction I. The influence of various amounts of palmitate on the labeling pattern was studied. Figure 5 shows the elution diagrams obtained with tryptic digests of samples of native BSA that have been trinitrophenylated with TNBS in solutions to which have been added 1 and 3 equiv of palm&ate per BSA, respectively. The labeling pattern obtained with defatted BSA is also shown. It is evident that the presence of palmitate causes a decrease in the labeling of Fraction I, probably by competition with TNBS for a binding site on the protein. The other frac-
AND
JOHANSSON
tions do not seem to be affected. The decrease in labeling is most pronounced when 3 equiv of palmitate has been added. This might] indicate t-hat the two strongest binding sites (7) for palmitate are not involved in the reaction with TSBS. Further fractionatjion of bhe various labeled fractions was performed using SESephadex and DEAE-cellulose chromatography. The purity of the final peptide preparations were tested by polyacrylamide gel electrophoresis (8). In Fig. 6 is shown the elution diagram obtained upon SE-Sephadex fractionation of Fract,ion I from gel filtrat,ion of a tryptic digest of native BSA labeled with an equivalent amount of TKBS. One main and one minor TNBS-labeled pept,ide is seen, here called I, and Ib . Peptide I, was obtained essentially pure as indicated by further fractionation on DEAE-cellulose and purity tests by polyacrylamide gel electrophoresis. The amino acid composition is given in Table I. From the estimated molecular weight of the pept’ide it is possible to give the estimated number of residues. The peptide obtained is fairly large, mol wt 12,500, and consequently the amino acid composition does not give very much indication of the nature of the labeled site. Therefore experiments were performed in order to break down the peptide further. Complete reduction with 0.1 nr mercaptoethanol in borate buffer pH 9.0 containing 1.0 % sodium lauryl sulfate followed by carboxymethylat’ion with iodoacetic acid and gel filtration on
Tube number
FIG. 6. SE-Sephadex chromatography elation diagram of Fraction I from gel filtration of tryptic digest of native BSA that had been treated with an equivalent amount of TNBS. Elution patterns: (-) absorbance at 254 nm, (-O-O-) absorbance at 350 nm.
BINDING
OF FATTY
ACID TABLE
AMINO ACID
COMPOSITIONS
Amino acid
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine % Cystine Valine Methionine Isoleucine Leucine Tyrosine
Phenylalanine CM-cysteine Total
TO SERUM I OF VARIOUS
I&
CI
11.3 2.1 -
10.0 0.8 -
0.7 -
0.7 -
18.0 5.9 2.0 15.6 9.0 0.1 14.1 3.4 4.0 -
17.0 4.3 1.0
0.1 -
0.1 -
13.2 6.8 -
1.0 -
12.3 -
1.0 -
3.0 2.1
-
6.5 3.7 3.0 4.3
1.0 0.9 -
106
88
5
0.10 A:“58 0.15
FIG. 7. Sephadex G-25 gel filtration elution diagram of chymotryptic digest of peptide I, Elution patterns: (-O-O-) absorbance at 280 nm, (--t-W) absorbance at 350 nm.
Sephadex G-50 yielded a labeled peptide that contained 18 amino acid residues less than the original peptide as shown in Table I. Chymotryptic digestion of the pure peptide I, was then tried. About 4 mg of peptide was digested with 0.5 mg of chymotrypsin for 2 hr at pH 7.8 and room temperature. Figure 7 shows the Sephadex G-25 gel filtration elution diagram of the digest. The double peak appearing last in the diagram was subjected to SE-Sephadex chromatography and resolved into two components called C I and C II. The results of the amino acid analyses,
PEPTIDES
Residues/mole of peptide c II II,
Red. CM I,
1.9 6.7 5.7 4.0
433
ALBUMIN
1.0 0.1 1.0 0.9 4
IL
III,
10.5 3.4 1.1 15.5 5.8 3.9 16.7 6.0 2.9 9.3 7.2 3.6 1.0 1.0 7.9 3.7 4.2 -
8.6 1.8 1.3 12.1 5.2 2.8 12.4 6.2 2.0 6.0 5.2 2.5 1.0 1.1 7.2 1.8 4.1
107
82
7.9 1.5 0.2 8.0 2.2 4.2 9.7 3.6 1.0 2.6 5.3 2.0 3.6 6.0 1.6 1.1 63
which are given in Table I, clearly show that the two fractions are derived from the same part of peptide I, . They contain the same amino acids but C II contains one leucine residue less. N-t’erminal analysis using dansylation at, pH 8.5 followed by acid hydrolysis of the dansylated peptide and identification of the dansyl amino acids by thinlayer chromatography on polyamide sheet’s (9) showed that leucine was the N-terminal amino acid in the pentapeptide and alanine the N-terminal in the smaller peptide. Peptic digestion of peptide I, yielded a TK’BSlabeled tripeptide containing glutamic acid, alanine, and lysine. Alanine was the N-terminal amino acid. By digestion of this peptide for 5 days with carboxypeptidase A at pH 8.5 and at 5” followed by gel filtration on Sephadex G-25 it was possible to isolate a peptide having the same Rf value on paper chromatography as the dipept,ide Ala-Glu. These data only leave one possible sequence for the pentapeptide, the one shown below. TNP leu-ala-glu-lys-tyr
Peptide Ib was not obtained
completely
434
ANDERSSON.
BRANDT,
0.2 A 254 350
Tube number FIG. 8. SE-Sephadex chromatography elution diagram of Fraction II from gel filtration of tryptic digest of a native BSA sample that had been treated with an equivalent amount of TNBS. Elution pat,terns: (---) absorbance at 254 nm, (-a-@--) absorbance at 350 nm.
pure but the results of the amino acid analysis indicate that it is identical with the peptide II, described below. In Fig. 8 is shown the elution diagram of the SE-Sephadex fractionation of Fraction II from gel filtration of tryptic digest of trinitrophenylated BSA. It contains three major and two minor TNBS-labeled peptides. Further fractionation of the three main peptides by DEAE-cellulose chromatography yielded the pure peptides. Their amino acid compositions are shown in Table I. The data indicate that peptide IIt, is derived from peptide II, . A peptide containing 25 amino acids appears to be split off in the conversion of II, to Iii, . The peptides II, and 16, are not related to peptide I, . The molecular weights as calculated from the amino acid compositions are 12,100 for peptide II, and 9,200 for IIb . The value for II, is somewhat hight compared to the value 8,500 obtained from the gel filtration data. It has to be remembered, however, that molecular weight estimations by gel fiItration in ordinary buffer solutions can give erroneous values when the type of conformation of the protein or peptide is different from that of the proteins or peptides used for the calibration curve. In this case it seems clear that the molecular weights obtained from the amino acid analysis data are considerably more reliable than those obtained by gel filtration. The small size of the fragments, the purity, and the good amino acid analysis data ob-
AND
JOHANSSON
tained make Dhe estimation of number of residues fairly safe. Peptide II, is not related either to peptides II, and IL, or to peptide I, . It contains three disulfide bonds and has a molecular weight of 7200. It probably elutes on the rear side of the Fraction II peak. This peak is rather broad. SE-Sephadex chromatography of Fraction III resolved it int.0 five of six minor peaks. It was therefore decided not to study this fraction further, especially as it contains a rather small part of the total TNBS label. SE-Sephadex chromatography of Fraction IV yielded one main TNBS-labeled peptide and traces of two others. Further fractionation on DEAE-cellulose revealed another minor TNBS-IabeIed peptide and also separated t,he main part of a tyrosine-containing peptide from the main TNBS-peptide of this fraction. Amino acid analysis indicated the composition of the TNBS-peptide to be one asparagine, one lysine, and two alanine residues. Binding studies. The question of whether TNBS is bound to BSA before it reacts is somewhat difficult to test directly because the reaction interferes with the binding measurements. This was accomplished, however, by making use of special conditions as described in the hilaterials and Methods section. Ultrafiltration of various TNBS-BSA mixtures through dialysis tubing at 5” and p-5 e
I,
3
2
1
L
1
2
3
bJ5
FIG. 9. Scatchard plots of binding data obtained by ultrafiltration experiments where the binding of TNBS to native and trinitrophenylated BSA was studied at 5” in 0.02 M phosphate buffer pH 7.2. Native BSA (O), trinitrophenylated BSA containing 3.0 TNP groups/BSA (A).
BINDING
OF FATTY
ACID
pH 7.2, where the reaction between TNBS and BSA is slow, followed by measurements of the TNBS concentrations in the earlier fractions of the filtrate, yielded values from which the binding curve shown in Fig. 9 could be calculated. Under these conditions TNBS and BSA react only to a very limited extent during the lo-15 min employed for the ultrafiltration. The binding curve obtained indicates that there are two strong (K = 1.7 X lo5 &x-l) and three weak (K = 1.5 X lo4 M-~) binding sites. Some measurements were also performed at pH 6.S and 7.5. The values obtained were about the same as those obtained at pH 7.2. Experiments were performed in which the binding of TNBS to :BSA samples that had been reacted with various amounts of TNBS was studied. It was found that the Drinitrophenylation altered the binding properties of BSA considera,bly but that one strong binding site seemed.to be unaffected. Figure 9 shows the binding curve obtained with a BSA sa,mple which had been reacted with 3 equiv of TNBS per BSA. Further experiments with BSA reacted with various amounts of TNBS showed that most binding sites were affected except one. This indicates that there is one strong binding site for TNBS which does not contain any reactive amino group. In order to obtain information on why TNBS is bound to BSA, binding studies were made on some substances related to TNBS. The substances studied were picric acid, TNP-glycine, and trinitrobenzene. The technique used to determine the binding to BSA was the ordinary equilibrium dialysis. The binding data are given in Table II. Studies at pH 7.0 and 5” with picric acid showed about the same binding curve as at pH 8.0. These binding studies were limited to the concentration range of 5 X 1O-7-1O-4 M thereby neglecting low affinity binding. Some experiments were performed in which the effect of caprylate on the binding of TKP-glycine to native and trinitrophenylated BSA was studied. Caprylate competed, in fact, with TNP-glycine both for the strong and weak binding sites in native BSA but the competitive effect was stronger with trinitrophenylated BSA. From
TO SERUM
435
ALBUMIN TABLE
II
BINDING OF VARIOUS TRINITROPHENYL DERIVATIVES TO BSA AS STUDIED BY EQUILIBRIUM DIaLYSIS IN 0.02 M PHOSPHATE BUFFER PB 8.0 AT 5” Substance
Trinitrobenzene Picric acid TNP-glycine TNBSa D Determined
_____ nl
2
Kl(K’)
5 x
n2
Kz(@)
103
2 1.2 x 106 2 6 X lo5 2 1 1.7 x 106
2 2 3
by ultrafiltration
at pH 7.2.
105 106 1.5 x 104
the competitive effect of caprylate on the binding of TNP-glycine the association constant of caprylate for binding to the strong site that remains unaffected by the trinitrophenylation could be estimated to be about 2 X lo5 M-~. The binding of caprylate to other sites as obtained from the results shown below has been taken into account in the calculation. To study if the trinitrophenylation did affect the binding of fatty acids, experiments were performed in which the binding of caprylate and palmitate to native and trinitrophenylated BSA was compared. Equilibrium dialysis at 5” with 14C-labeled caprylate in 0.02 11 phosphate buffer pH 8.0 yielded binding curves which showed the presence of 1.6 moderately strong binding sites with K = 3.5 X lo5 M-I in native nondefatted BSA. Charcoal-defatted BSA showed 2.0 binding sites with K = 3.1 X lo5 b1-l. Trinitrophenylated BSA containing 3.0 trinitrophenyl groups per BSA shoxved the same number of strong binding sites and the same association constants as native BSA but less low--affinity binding of caprylate. Binding studies on palmitate and ot,her fatty acid anions of that size or bigger are usually performed in two-phase systems with one water phase and one hydrocarbon phase (7, 10). This is done in order to overcome the low solubility of these fatty acids in water and to be able to study a broad range of fatty acid concentrations. A disadvantage with this method is that the presence of the hydrocarbon solvent may introduce artifacts. There are indications of conformational
436
ANDERSSON,
BRANDT,
AND JOHANSSON
albumin obtained from AB Kabi, Stockholm, containing 0.92 equiv of bound fatty acids. The number of strong binding sites was 0.9 and the association constant was 3.1 X lo6 JI-’ which is only a slightly higher value than that obtained with BSA. The reason for the different results obtained in this study and the study by Goodman (7) is evidently to be found in the different methods used to determine the binding. Changes in conformation and/or motility. I To study whether changes in conformation 1 2 c and/or motility (13) occur on binding to or FIG. 10. Binding of palm&ate to native and modification of BSA the rate of enzymatic trinitrophenylated BSA as determined by equilibhydrolysis of BSA was studied. This is a rium dialysis in 0.02 M phosphate buffer pH 8.0 very sensitive method for detecting changes and 5”. Native BSA (O), trinitrophenylated BSA in conformation and motility (14, 15). The containing 3.0 TNP groups/BSA (A). hydrolytic enzymes used were trypsin and chymotrypsin. The rate of hydrolysis was changes of BSA caused by heptane dissolved followed by titration with 0.02 M NaOH soluin water (11). Another difficulty with the tion in an automatic titrator (Radiometer, method is to estimate the extent of dimerizaCopenhagen). The ratio mg enzyme/mg BSA tion of the fatty acid anions in the aqueous was l/100, the temperature 25”, and the pH phase (12). Because of the uncertainties S.O. The hydrolysis curves obtained were involved in the two-phase method, it was close to straight lines after 1 or 2 min after decided to use the ordinary equilibrium the initiation of the hydrolysis. The results dialysis method. This, however, limited the obtained are shown in Table III. It is clear study to high affinity binding. The binding that the pattern obtained is the same both curve obtained with native BSA at 5’ in 0.02 with trypsin and chymotrypsin. All subM phosphate buffer pH 8.0 is shown in Fig. stances studied do affect the rate of digestion 10. It indicates the presence of 1.3 strong binding sites with K = 2.2 X lo6 111-l.The but there seems to be no simple relation between the strength of the binding and the binding data obtained with trinitrophenyrate of digestion. The rate of hydrolysis of lated BSA containing 3.0 trinitrophenyl trinitrophenylated BSA is very low. The exgroups per BSA are about the same as with planation is probably that the covalent native BSA in the high affinity binding coupling of TNBS provides for a more effirange, but there seems to be a difference with cient stabilization. The fact that a few lysine respect to the further binding of palmitate. residues have been modified probably makes Unfortunately the limited concentration range attainable for the studies with the the rate of tryptic digestion lower, but this effect cannot be large as chymotryptic digespresent method does not permit a quantitation gives about the same results. tive evaluation of this further binding of palmitate. However, the data obtained DISCUSSION clearly show that the trinitrophenylation The kinetics of the reaction between does affect this type of binding. The association constant for the binding of TNBS and BSA at low concentrations of palmitate to the two strongest sites in BSA TNBS is close to first order. This is perhaps somewhat surprising because one might exobtained in this study is much lower than that for human serum albumin (K = 6 X pect a second-order rate behavior of this bimolecular reaction. If, however, TNBS is 10’ M-‘) as determined by the two-phase partition method (7). To clarify the reasons rapidly bound to BSA and reacts in the comfor this difference, some measurements were plexed form, a first-order behavior would be performed on a sample of human serum expected. That rapid binding of TNBS to
BINDING
OF FATTY
ACID
437
TO SERUM ALBUMIN
TABLE III BSA FGLUTIONS CONTAINING VARIOUS ADDED SUBSTANCESO
DIGESTION RATES OF NATIVE AND MODIFIED
Sample
Digestion rate with trypsin bmole/min) Digestion rate with chymotrypsin (I*mole/min)
BSA
TNP-BSA (3.0TNP/BSA)
~s$c$ gibp;ylate BSAcOT6 pd$de
BS.4 $.gpis
0.114
0.026
0.086
0.098
0.076
0.158
0.047
0.100
0.118
0.087
a Two per cent BSA solutions were digested with 1 to 100 of hydrolytic enzyme in 0.1 M N&l pH 8.0 and 25”. The digestion was followed by titration with 0.02 M NaOH in an automatic titrator.
BSA actually occurs is evident from the binding studies on the TNBS-BSA system. From these results and the observation that TNBS treatment blocks most of the binding sites for TNBS, it seems rather safe to conclude that at, low TNBS/BSA ratios the main part of the reaction occurs between TNBS bound to various binding sites and amino groups present in the vicinity of these sites. Reduction and carboxymethylation of BSA probably destroys the binding sites for TNBS and should accordingly lower the rat’e of the reaction with TNBS. This is in fact what is observed, viz., that although the total number of available amino groups is larger in reduced and carboxymethylated BSA, the initial rate of the reaction is slower than with native BSA. The studies on binding of substances similar to TNBS give some indications of the nature of the binding sites. First, it seems fairly obvious that picric acid, TNP-glycine, and trinitrobenzene are bound to binding sites that also1bind TNBS. The experimentally determined association constants show that the binding of trinitrobenzene is much weaker than the binding of the other substances. This is probably due to the fact that the other substances are ions and electrostatic interactions with ionic groups of the opposite charge on the protein contribute to the binding. Calculation of the free-energy change upon binding of trinitrobenzene to BSA from the apparent association constant yields the value 4.7 kcal/mole. This change in free energy results from the transfer of trinitr0benzen.e from water to binding sites in the protein. The binding of trinitrobenzene to BSA shou1.d probably be regarded as a
acid
at
pure hydrophobic bonding, and consequently it is possible to use the value 4.7 kcal/mole for the hydrophobic contribution in the binding of picric acid, TNBS, and TNPglycine. It is, however, possible that some hydrogen bonding may contribute to some extent to the binding of the trinitrobenzene to BSA. The electrostatic contribution to the freeenergy change upon binding of picric acid, TNBS, and TNP-glycine can be estimated by subtracting the value for the hydrophobic contribution from the total change in free energy on binding of these substances. This gives values in the range of 2.0-3.0 kcal/ mole. The value for picric acid is especially high. The conclusion that follows from these calculations and considerations is that hydrophobic and to a lesser degree electrostatic interactions are mainly responsible for the binding of picric acid, TNBS, and TNPglycine. This is very similar to the situation with binding of long-chain fatty acid anions (16) to serum albumin. The study has been limited to the pH range 7.0-8.4 thus being mainly concerned with binding at pH values close to that of the blood. In the studies on the kinetics of the reaction between TNBS and BSA it was found that the presence of fatty acids could affect the rate of the reaction in various ways depending on the size of the fatty acid added. Long-chain fatty acids such as palmitate decreased the rate of the reaction. The reason for this is probably that pahnitate is bound to one or several binding sites which may also bind and react with TNBS. The rapid reaction between TNBS and BSA at low
438
ANDERSSON,
BRANDT,
TNBS/BSA ratios depends on the binding of TNBS t’o binding sites containing amino groups, and it is quite clear that the rate of the reaction must diminish if a substance that is bound to one or several of these sites is added. This explanation is also in agreement with the observation made in connection with the labeling experiments that the addition of pahnitate caused a decrease in the degree of labeling of Fraction I. The increase of the reaction rate caused by caprylate is somewhat more difficult to explain. However, remembering the fact that there is one strong TNBS binding site that does not contain any reactive amino group it is possible to find a reasonable explanation. If this nonreactive TNBS binding site also binds caprylate, the addition of caprylate to the solution would increase the concentration of free TNBS because of the competitive binding. If caprylate is not at all, or weakly, bound to the reactive TNBS binding sites, this would result in an increase in the rate of the reaction between TNBS and BSA. Further support for this explanation is obtained from the fact that on increasing the TNBS/BSA ratio the rate-increasing effect of caprylate disappears and it becomes slightly rate decreasing. This indicates that caprylate is in fact weakly bound to the reactive TNBS binding sites. The results of the studies on competitive binding of TNPglycine and caprylate bo native and TNBStreated BSA are in agreement with the explanations suggested above. The binding of TNBS and related substances to serum albumin shows many similarities to the binding of fatty acid anions. The studies on the labeling of binding sites with TNBS shows that one of the most reactive sites is involved in the binding of palmitate. The observation that defatting increases the reactivity of this site might be taken as an indication that it is one of the two strongest binding sites for palmitate. However, the results from the studies on the binding of palm&ate to native and trinitrophenylated BSA show that the two strongest binding sites are not involved, but that probably one of the moderately strong binding sites is blocked by the trinitrophenylation. It thus appears as if the reactive site in the
AND JOHANSSON
Fraction I peptide is one of the moderately strong binding sites for long-chain fatty acids. The results of the labeling experimenbs with various amounts of palmitate present in the reaction mixture are also in agreement with this conclusion. The amino acid sequence around the reactive lysine shows some interesting features. The lysine residue has on one side a glutamic acid residue with which it probably interacts and on the other side a tyrosine residue. The presence of this tyrosine residue immediately adjacent to the lysine should probably be correlated with the observation (16) that the binding of fatty acids induced shifts in the uv-spectrum of BSA which are characteristic for changes in tyrosine environment. In connection with studies on the binding of 1-anilino-S-naphthalene sulfonic acid (ANS) to BSA, Anderson and Weber suggested (17) that ANS was bound to clefts between various globular parts of BSA. These globular parts have been called “subunits,” and there are indications from several types of measurements (E-20) that the peptide chain of serum albumin is folded so as to constitute three or four globular parts (20), the subunits. It is an attractive hypothesis that binding of fatty acid anions should occur to clefts between subunits. If the inner parts of the clefts are hydrophobic and some lysine or arginine residue is situated on the outer part of the cleft a very favorable situation for binding would be present. Hydrophobic and electrostatic bonding would be the main binding forces as is the case on binding of long-chain fatty acids (16) and detergents (21). The subunit hypothesis suggests that there is some degree of flexibility between the subunits. This can explain several different observed effects of fatty acids and detergents on serum albumin. The binding of a fatty acid anion to a cleft between two subunits would certainly diminish the freedom of movement of the two subunits versus each other. Another effect closely linked to this is the stabilization of the whole protein molecule obtained by not exposing the hydrophobic parts of the binding site to water. These two effects, the stabilization and the
BINDING
OF FATTY
ACID
decreased motility, are in agreement with the observations that long-chain fatty acids stabilize serum albumin against heat denaturation (22) and that binding of fatty acid anions and similar substances makes the albumin less susceptible to attack by proteolytic enzymes (23). Measurements of optical rotation have shown (24) that the binding of small amounts of detergents does affect the conformation of serum albumin. It is possible that binding to the clefts between the subunits is a general mechanism for the binding of various substances to serum albumin. The conformation and motility changes can also be put in relation to some observations made on the isoelectric focusing behavior of serum albumin, in which it was found that the binding of fatty acid anions greatly influenced the p1 of the protein (25). The p1 diminished from about 5.8 to 4.7 on binding of fatty acid anions. A possible explanation is that the conformational changes taking place upon binding, among other things, also buries some amino groups. Indications for the presence of buried amino and carboxyl groups in native BSA have been obtained by Avruch et al. (26) who found that acetimidation of the lysine residues in BSA altered the charge and the carboxyl titration behavior of BSA. Vijai and Foster (27) have suggested that pairs of unionized carboxyl and. amino groups are situated in the clefts between the subunits of BSA. The main features of the binding mechanism suggested are indicated below using a simplified section model of serum albumin with two subunits. Hydrophobic areas are shaded.
Defat.ted st,ate High motility Low stability
Fatty acid containing state Low motility High stability
TO SERUM ALBUMIN
439
One of the assumptions made in the derivation of the Scatchard equation (5, 6) for binding to multiple sites is that all sites behave completely independently of each other. This is probably not the case in the binding of fatty acids and detergents to serum albumin. Both the observed changes in conformation and motility and the nature of the subunit model strongly indicate that binding to one or several sites has an influence on the binding to the remaining sites. From this it follows that the values obtained for the number of binding sites and binding constants must be regarded as somewhat uncertain. The increase in TNBS labeling of Fraction I when defatted BSA is used may be due t’o general conformation or motility changes upon defatting which also affect sites not containing fatty acid originally. ACKNOWLEDGMENTS We thank Dr. C-G Rosen for valuable discussion and criticism of the manuscript. The study has been supported by grants from Swedish Board for Technical Development. REFERENCES 1. 2.
GOLDFARB, GOLDFARB,
A. R., Biochemistry 6, 2574 (1966). A. R., Biochim. Biophys. Acta 200,
1 (1970). 3. DOLE, V. P., J. Clin. Invest. 35, 150 (1956). 4. CHEN, R. F., J. Biol. Chem. 242, 173 (1967). G., SCHEINBORG, I. H., AND 5. SCATCHARD, ARMSTRONG, S. H., J. Amer. Chem. Sot. 72, 535, 540 (1950). 6. SCATCHARD, G., COLEMAN, J. B., AND SHEN, A. L., J. Amer. Chem. Sot. 79, 17 (1957). 7. GOODMAN,D. S., J. Amer. Chem. Sot. 60, 3892 (1958). 8. RAYMOND, S., Ann. N. Y. Acad. Sci. 121, 350 (1964). 9. WOODS, K. R., AND WANG, K.-T., Biochim. Biophys. Acta 133, 369 (1967). 10. ARVIDSSON, E. O., Small molecule-protein interactions, Thesis, Studentlitteratur, Lund (1965). Il. ALFS~N, A., C. R. Trav. Lab. Carlsberg 33, 415 (1963). 12. MUKERJEE, P., J. Phys. Chem. 69, 2821 (1965). 13. TANIUCHI, H., MORAVEK, L., AND ANFINSEN, C. B., J. Biol. Chem. 244, 4600 (1969). 14. MARKUS, G., Proc. Nat. Ad. Sci. U. S. A. 64, 253 (1965).
440
ANDERSSON,
BRANDT,
15. MARKUS, G., MCCLINTOCK, II. K., AND CASTELLINI, B. A., J. Biol. Chem. 242, 4402 (1967). 16. REYNOLDS, J., HERBERT, S., AND STEINHARDT, J., Biochemistry 7, 1357 (1968). 17. ANDERSON, S. R., AND WEBER, G., Biochemistry 8, 371 (1969). 18. BLOOMFIELD, V., Biochemistry 6, 684 (1966). 19. WEBER, G., AND YOUNG, L. B., J. Biol. Chews. 289, 1424 (1964). 20. PEDERSON, D. M., AND FOSTER, J. F., Biochemistry 8, 2357 (1969). 21. RAY, A., REYNOLDS, J. A., POLET, H., AND STEINHARDT, J., Biochemistry 6, 2606 (1966).
AND
JOHANSSON
22. BOYER, P. D., LUM, F. G., BALLOU, G. A., LUCK, J. M., AND RICE, R. G., J. Biol. Chem. 162, 181 (1946). 23. KLEPPER, J. A., JR., AND CANN, J. R., Arch. Biochem. Biophys. 108, 531 (1964). 24. REYNOLDS, J. A., HERBERT, S., POLET, H.! AND STEINHARDT, J., Biochemistry 6, 937 (1967). 25. VALMET, E., in “Protides of Biological Fluids” (H. Peeters, ed.), Vol. 17, p. 443 (1969). 26. AVRUCH, J., REYNOLDS, J. A., AND REYNOLDS J. H., Biochemistry 8, 1855 (1969). 27. VIJAI, K. K., AND FOSTER, J. F., Biochemistry 6, 1152 (1967).