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Application of spectrophotometric methods to the determination of protein bound to agarose beads
ANALYTICAL
BIOCHEMISTRY
Application
66,
556-567 (1975)
of Spectrophotometric the Determination Bound
Methods
to
of Protein
to Agarose
Beads
REGINE KOELSCH, J. LASCH, IRMGARD MARQUARDT, AND H. HANSON Physiologisch-chemisches Institut der Martin-Luther-Universitat, 402 HallelSaale, Hollystrasse l( GDR Received September 20, 1974; accepted January 16, 1975 Quantitation of the amount of leucine aminopeptidase and trypsin covalently attached to Sepharose 6B using the CNBr method was accomplished by five independent methods: protein balance (estimation of the amount of protein not recovered in the final washings), amino acid analysis, modified Lowry method, spectrophotometric and fluorophotometric analysis, the latter two being practicable with the undestroyed gel beads. These methods adapted to the special conditions in particulate enzyme systems were investigated in detail and the results compared. The last three methods were found to be simple, sensitive and highly reproducible.
Attachment of enzymes to insoluble carriers has become a frequently used technique in many laboratories. Especially, the coupling of proteins to polysaccharide supports by the CNBr method is now a very common procedure. The exact determination of the amount of bound protein is highly important for a number of reasons. It is particularly necessary if one has to measure how the enzymic activity has been modified by immobilization (e.g., partial inactivation) or if one has to estimate kinetic parameters of the bound enzyme in comparison with the free enzyme in solution. Accurate protein determinations are also indispensable for the proof of enzyme leaching from the support. If one uses CNBr-activated Sepharose or Sephadex gels, protein evaluations from the nitrogen content of the product are impossible. The most frequently used method is quantitative amino acid analysis after acid hydrolysis of the dried protein-carrier conjugate (l), which seems to have acquired widespread use as standard procedure (2). Another often employed method is to estimate the bound protein by protein “balance”, i.e., to calculate the difference between the amounts of protein added and those found in the supernatant fluid and washings after the coupling reaction. Havekes and co-workers (3) have reported the quantitation of Se556 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
ANALYSIS
OF
AGAROSE-BOUND
PROTEINS
557
pharose-bound glutamate dehydrogenase by a modified Lowry (4) procedure. Spectrophotometric measurements were carried out by Failla and Santi (5) after acid solubilization of the agarose matrix in order to quantitate ligands used in affinity chromatography. To our knowledge, direct sprectrophotometric methods with the intact gel beads have not been used up to now, probably because many workers believe that the insoluble particles interfere with the measurements. By employing special geometric arrangements, we can show, however, that it is possible to measure the absorbance at 280 nm without destruction of the enzyme-carrying particles. It is the aim of our investigation to find out reproducible and accurate methods for protein determinations of matrix-bound enzymes, methods that are easily and quickly handled. For this purpose we compare protein determinations achieved by amino acid analysis after acid hydrolysis with the “balance” method, with a modified Lowry procedure and with a newly developed spectrophotometric method. Moreover, fluorescence intensity measurements using the front-viewing technique are included in the comparison. MATERIALS
AND
METHODS
Mnterials Sepharose 6B was a product of Pharmacia Fine Chemicals, Uppsala, Sweden. Cyanogen bromide was purchased from PH “Dr. Th. Neubauer,” Erfurt, GDR; twice crystallized trypsin from Serva, Heidelberg, GFR. Leucine aminopeptidase once crystallized from bovine eye lenses was routinely prepared in our institute (6). Amberlite IR 120-X8 was purchased from Rohm und Haas, Darmstadt, GFR, and Dowex 5OW-X4, p.a., 50-100 mesh, from Serva, Heidelberg, GFR. Activation
of Sepharose 6B and Coupling
Procedure
Sepharose 6B was thoroughly washed on a glass funnel and brought to twice its original volume by addition of water. This suspension was used for all activations. One milliliter of this mixture had, after drying under reduced pressure over PZ05, an average weight of 18.7 mg. For the activation procedure we took 1O-ml portions of the Sepharose suspension. Activations were carried out at 15°C using a final CNBr concentration of 20-33 mglml. An adjusted pH of 10.5 was attained by automatic addition of 4 N NaOH using a TTT lc Titrator (Radiometer. Copenhagen, Denmark), and the activation was allowed to proceed at this pH for 6 min. After washing the activated gel with water and 0. I M NaHCO, on a G-3 glass filter, the gel was brought to 10 ml with 0.1 M
558
KOELSCH
ET
AL.
NaHC03. The enzyme solution, 2 ml, about IO-20 mg/ml, was added. The mixture was gently shaken overnight. Thereafter it was filtered and the gel washed sequentially with 0.1 M NaHCO,, 1 M KC1 and H20. Then the gel was brought to a final volume of 10 ml by addition of 0.1 M KC1 and stored at 4°C. Protein
Determination
Methods
Method A: Protein balance. The amounts of protein conjugated on the gel particles were estimated from the difference between the initial amounts of protein added and those found in the pooled filtrate and washings. Protein concentrations were measured by a micro Lowry method (4,7). Method B: ModiJied Lowry reaction. The Lowry reaction (4,7) was carried out with 200 ~1 of the enzyme-Sepharose 6B suspension. During the reaction time (140 min) the mixture was gently shaken at room temperature and centrifuged before absorbance readings of the supernatant fraction were taken. Blanks were composed of equal amounts of unactivated Sepharose 6B suspension. Method C: Ultraviolet spectraphotometry. Measurements were carried out in a Unicam SP 800 Spectrophotometer (Pye Unicam Ltd., Cambridge, England) using the constant-temperature cell-housing for second sample-position SP 871 (directly in front of the photomultiplier). The gel was gravity packed in a 2-mm light-path cuvette and unactivated Sepharose 6B placed in the reference beam. The absorbance at 280 nm was chosen for the calculation of protein amounts using the same molar absorption coefficient as for the enzymes in solution (3.26 X lo5 M-‘cm-’ for leucine aminopeptidase (8) and 3.58 X lo4 M-‘cm-’ for trypsin (9)). We define “concentration” operationally as moles of enzyme per liter of packed gel. This concentration can be easily converted to milligrams of bound enzyme per gram of dry-weight support knowing that 1 ml of gravity-packed gel is equivalent to 48 mg dry weight. This relationship was reproducibly obtained from the dry weight of 1 ml of Sepharose 6B suspension and its sedimentation ratio (i.e., total volume: volume of the settled gravity-packed gel beads). Method D: Amino acid analysis. Samples of Sepharose-bound enzymes were washed with distilled water and dried in vacua over phosphorous pentoxide. Acid hydrolysis with 4 N HCl was carried out in heavy-walled tubes under exclusion of oxygen, using weighed amounts of polysaccharide-enzyme conjugates (about 30 mg) or mixtures of dried Sepharose with protein, and 2 ml of acid, with 1.25 &moles norleutine as an internal standard. Before sealing, the tubes were frozen at -72 “C, evacuated, twice warmed in a water bath and again frozen and evacuated. Hydrolysis conditions: Temperature 110 “C, hydrolysis time
ANALYSIS
OF
AGAROSE-BOUND
PROTEINS
559
24 hr. Hydrolyzates were black-colored and cloudy and had to be purified before amino acid analysis by pretreatment on an ion-exchange column. After removal of the hydrochloric acid in a rotary evaporator, the samples were dissolved in 2 ml of 0.02 N HCl, applied either on a column of Amberlite IR 120-X8 and eluted with 25 ml of 1 M ammonia or on a column of Dowex 5OW-X4 and eluted with 25 ml of 10% pyridinelwater (v/v). The dimensions of both columns were 1.0 X 7.0 cm. The residue, taken to dryness three times on the rotary evaporator, could be redissolved in buffer, pH 2.2, conveniently diluted and filtered through hard filter paper. The recovery of amino acids during this purification procedure was proved in separate experiments. Amino acid analyses were performed with an Amino Acid Analyzer Unichrom (Beckman, Munich, GFR) according to the method of Spackman et uf. (10). The protein content was calculated from the estimated amounts of the amino acids aspartic acid, alanine, valine, leucine, and phenylalanine and the separately determined amino acid composition of the enzyme preparations employed for the immobilization. Method E: Fluorescence intensity measurement. Fluorescence emission measurements were done in front-surface viewing geometry with the Hitachi automatic recording fluorescence spectrophotometer, Model 204 (Hitachi, Ltd., Tokyo, Japan). The Sepharose-enzyme conjugates were gravity packed into l-cm2 cuvettes and the sample excited at 295 nm. Blanks consisting of packed Sepharose 6B showed no measurable fluorescence emission at the amplification chosen. Emission spectra of carrier-enzyme conjugates differing in their protein content were recorded and the areas under the recorder tracings measured by a planimeter. RESULTS
We have chosen leucine aminopeptidase (LAP), an oligomeric ( 11) enzyme with a molecular weight of 326,000 (12) and the monomeric trypsin with a molecular weight of 23,800 (13) as model enzymes. The results of protein determinations by methods A, B and C for Sepharose 6B-bound LAP and trypsin are given in Table 1. Method A must be regarded as rather inaccurate, especially in cases where small volumes of washings have to be handled or if the amount of bound protein is small. Nevertheless, one can see from Table 1 that there is, in general, a satisfactory agreement with the results obtained by other methods. Method B seems to be a very simple and accurate method with high reproducibility. For one sample of Sepharose-bound protein, determined three times on different days and after storing at 4°C between the es-
560
KOELSCH TABLE
COMPARISON
OF AMOUNTS
BOUND
PROTEIN
A method Milligrams a
AL.
1 DETERMINED
BY DIFFERENT
B Modified Lowry procedure
“Balance”
Sample
ET
Moles 10’6
Milligrams a
Moles 10’6
METHODS
C Spectrophotometry Milligrams a
Moles 1076
Sepharose 6B-LAP
32.1 49.7 48.2 12.8
1.0 1.5 1.5 0.4
32.8 46.7 41.7 13.4
1.0 1.4 1.3 0.4
29.8 42.4 43.5 14.2
0.9 1.3 1.3 0.4
Sepharose 6B-trypsin
45.5 44.4 83.0 33.7
19.2 18.7 35.0 14.2
38.5 47.1 68.0 39.8
16.2 19.8 28.6 16.6
44.0 47.4 65.0 34.6
18.5 19.9 27.3 14.5
’ Milligrams of enzyme per gram of dried support. * Moles of enzyme per gram of dried support.
timations, we found the following values: 0.59 mg/ml; 0.58 mg/ml; 0.58 mg/ml. At first we attributed the accuracy and reproducibility of this method to quantitative detachment of the protein from the carrier by the prolonged treatment of the suspension at alkaline pH. Appropriate reference experiments revealed, however, that the alkaline medium detached no more than 80%,of the bound protein from the gel beads. A quantitative estimation of bound protein thus necessitates treatment of the enzyme-loaded carrier particles with Folin’s reagent. This led us to the conclusion that the reduced phosphomolybdate complex diffuses freely out of the support. Method C is easy to handle. Gravity packing of the cuvettes is a bit time-consuming (it takes about 2 hr), but this is compensated for by the rapid and convenient measurement. Figures 1 and 2 show the uv spectra of Sepharose-bound leucine aminopeptidase and trypsin in comparison to the free enzymes in solution. The spectra of the bound enzymes are very similar to those of the free ones. It is only in the shorter wavelength region that the spectrum becomes distorted by the background absorption and light scattering of the polysaccharide carrier. Absorption at 280 nm can be read easily. In order to prove the validity of the Lambet-t-Beer law, absorbances were plotted against the amount of protein (LAP) attached to the carrier (Fig. 3). From the resulting straight line we calculate the same absorption coefficient (see Methods) as for the dissolved enzyme. The same holds true with trypsin. This finding
ANALYSIS
OF AGAROSE-BOUND
o.0
561
PROTEINS
0 wavelength
nm
FIG. 1. Ultraviolet spectra of soluble and insolubilized leucine aminopeptidase. I. I.AP dissolved in water; concentration, 1.63 X ~O+M; reference: water. 2, LAP covalently bound to Sepharose 6B. gravity packed: reference: Sepharose 6B: light path. 0.7 cm.
wavelength
nm
FIG. 2. Ultraviolet spectra of soluble and insolubilized trypsin. 1. Trypsin dissolved in IO-"M HCI; concentration, 1.04 X IO-'M: reference: ~O-"M HCI. 1, trypsin covalently attached to Sepharose 6B, gravity packed in a 0.2-cm light path cuvette; reference: unmodified Sepharose 6B.
562
KOELSCH
protein 3 106x
operational
ET
AL.
amount
tmg/g) 6
concentration
9 CM 1
3. Proof of the validity of the Lambert-Beer law for Sepharose-bound leucine aminopeptidase. Sepharose-bound LAP suspensions were pipetted into 0.2-cm light path cuvettes, gravity packed and the absorbance read at 280 nm. Reference: unmodified Sepharose 6B. Amounts of protein were determined by the modified Lowry procedure and by the “balance” method. FIG.
together with the observation that many structural and functional properties of leucine aminopeptidase are not changed upon coupling by the BrCN method (14,15) seem to justify the use of the same molar absorbance coefficient for the free and attached enzyme. It is seen from Table 1 that protein amounts found by this method are in good agreement with those found by Methods A and B. It seems that Method D, which might be believed to yield the most accurate results, has never been evaluated thoroughly and compared with other, particularly destructionless methods. The well-known precision of this method with pure proteins may be lost in the presence of support material. In order to verify that acid hydrolysis in the presence of polysaccharide does not lead to high losses of amino acids, dried Sepharose was mixed with known amounts of leucine aminopeptidase and subjected to the same hydrolysis conditions as the soluble and insoluble enzyme. The results are summarized in Table 2. There is fairly good agreement between the values of the selected five amino acids, pointing to the fact
ANALYSIS
OF
AGAROSE-BOUND
563
PROTEINS
TABLE 2 AMINO ACID ANALYSES: REFERENCE AND CONTROL EXPERIMENTS 1=
2a
30
Amino acid
Ab
B”
Ab
B’
Aspartic acid Alanine Valine Leucine Phenylalanine
0.31 0.35 0.20 0.26 0.12
234 264 1.51 196 91
0.34 0.37 0.20 0.26 0.12
257 219 151 196 91
Ab
B’
Xd
X
X
X
X
X
0.25 0.12
187 91
0 1, Reference: 2.16 mg of leucine aminopeptidase; 2, 2.16 mg of LAP + 29.6 mg of Sepharose 6B, after acid hydrolysis followed by adsorption on a Dowex SOW-X4 column and elution with 10% pyridine; 3, 2.16 mg of LAP + 30.4 mg Sepharose 6B, after acid hydrolysis followed by adsorption on a Dowex 5OW-X4 column. and elution with 1 M ammonia. b Micromoles amino acid per milliliter of hydrolyzate. c Number of residues. d x, Overlapping peaks, estimation not possible.
that, at least for these amino acids, interference by the added polysaccharide material is negligible. In experiments where the hydrolyzed sample was eluted from the prewash column (either Amberlite or Dowex) by 1 M ammonia it was consistently found that the subsequent chromatography does not result in sharp peaks of aspartic acid, alanine and valine. These peak distortions are probably caused by undesired secondary hydrolysis products. With the emergence of the second buffer according to the standard procedure, (10) leucine and phenylalanine, however, emerged from the column as the usual sharp peaks. In these cases, the calculation of the amount of protein was based only on leucine and phenylalanine. These difficulties could be avoided in later experiments by employing a Dowex SOW-X4 prewash column and eluting with 10% pyridine. The results of amino acid analysis are summarized in Table 3 along with the values obtained by other methods. The finding that the amounts of bound protein as determined by amino acid analysis are consistently lower than those estimated by other methods may be explained by appreciable side-reactions of the derivatized carrier during acid hydrolysis. Interestingly, the hydrolysis side-reactions of derivatized and underivatized Sepharose seem to be quite different under otherwise identical conditions of hydrolysis as already evident by a difference in the color of the hydrolyzates. Method E utilizes the linear proportionality between fluorescence signal and fluorochrome concentration that holds for sufficiently small concentrations for which the usual exponential relation between measur-
Pretreatment
Sample
OF BOUND
value from “balance” method from Lowry method from spectrophotometry
PROTEIN
TABLE
3
26.81 28.79 17.27 21.11 11.52
A”
1.15 1.5 1.3 1.3
1.15 1.09 1.14 1.08 1.27
B* 8.52 8.52 4.26 7.1 2.84
A”
3.3 3.9 4.0 4.4
3.6 3.2 2.8 3.6 3.1
Bb
Dowex 50-X4, pyridine
13.68 16.72 7.60 9.12 4.56
A”’
Sepharose 6B-leucine
5.4 6.0 4.6
5.9 6.3 5.0 4.7 5.0
B”
5.9 9.0 6.5
X
5.7 6.1
X
x
X
B”
Amberlite,
11.15 5.58
XC X
A”
aminopeptidase
17.36
X
X
X X
A”
1.9 2.6 2.0
1.9
X
X
X X
Bb
“BALANCE”
ammonia
AS DETERMINED BY AMINO ACID ANALYSIS AFTER ACID HYDROLYSIS. THE MODIFIED LOWRY REACTION, AND THE SPECTROPHOTOMETRIC METHOD
UA, Micromoles of amino acid per gram of dried support. * B, Moles of enzyme per gram of dried support. COverlapping peaks, estimation not possible,
Mean Value Value Value
Aspartic acid Alanine Valine Leucine Phenylalanine
-
AMOUNT
THE
36.00 24.32 24.32 25.30 5.84
Afl
2.0 3.5 2.9 2.7
1.89 2.03 2.03 2.11 1.95
Bb
Dowex 50-X4, pyridine
Sepharose 6B-trypsin
METHOD,
.F
2
i
6 F
ANALYSIS
OF AGAROSE-BOUND
565
PROTEINS
1
J
3 lo6
x operational
6 concentration(M
9 1
FIG. 4. Measured areas under the fluorescence intensity spectra plotted against amounts of protein estimated by other methods. 0. Protein determined by spectrophotometry: Cl. protein determined by “balance” method. For conditions, see Methods.
able fluorescence intensity F and concentration, F = F,+ (1 - P-“.:~~~~~~“‘), simplifies to F = 2.303 F&cd, where F, = excitation energy, 4 = relative quantum efficiency, E = molar absorption coefficient (M-‘cm-‘), c= molar concentration, and d = thickness (cm). Figure 4 demonstrates that the linear correlation between fluorescence intensity and amount of enzyme bound can be readily used to quantitate insolubilized proteins. The areas under the emission spectra were measured in connection with the evaluation of apparent quantum yields. Intensity measurements at the wavelength of the maximum would yield the same linear correlation. The front-surface viewing technique allows even dense enzyme-carrier suspensions to be determined. DISCUSSION
Failla and Santi (5) described a number of methods for solubilizing derivatized agarose beads permitting quantitative spectrophotometric analysis of covalently attached ligands. Although this method seems to work satisfactorily when the solubilization is effected by 50% acetic
566
KOELSCH
ET
AL.
acid, we found that this procedure may run aground in a number of instances. First, at the usual degree of Sepharose activation by cyanogen bromide, generally not less than 20 mg of BrCN per ml, Sepharose-protein conjugates can not be dissolved completely by treatment with 50% acetic acid at 70°C in contrast to underivatized Sepharose 6B which gives a clear solution after the same treatment. This is readily attributed to cross-linking during the activation step. Second, the spectra of the supernatant portions of acetic acid-treated Sepharose-enzyme conjugates showed distortions probably due to light-absorbing decomposition products of the matrix or, more likely, of the introduced functional groups. Warming the gel beads in acidic or alkaline media confirmed the observations reported by the authors cited above (5) that solubilization in 0.1 M HCI (15 min, 100°C) and I M NaOH produced byproducts that prohibited a quantitative analysis of the spectra. It follows that only appropriately modified and solubilized Sepharose (a nonabsorbing compound like glycine may be coupled) is the correct substance to be used in the reference cuvette in order to blank out uncontrollable background absorbance. Third, the spectra of unmodified Sepharose set lower limits to the spectrophotometric measurement of the concentration of bound ligands. The concentrations encountered in experiments with immobilized enzymes are far below these limits. In an attempt to overcome these difficulties we tried successfully to adapt spectrophotometric and lluorophotometric assays to undestroyed Sepharose beads. One principal problem that arises is how to calibrate the different methods. We chose for calibration and comparison the amino acid analysis and the modified Lowry method, the latter giving highly reproducible results. In addition, the agreement between the values obtained by different and independent methods confirms the usefulness of the investigated methods. The finding that the amino acid analysis consistently affords values of the amount of bound protein that are too low seems to cast doubt on the usefulness of this method in the presence of high amounts of polysaccharide carrier. That this is not true with immobilized oligopeptides (R. Yost and A. Jarron, personal communication) may be explained by the less extensive side-reactions in the hydrolysis mixture. The lower limits for the determination of the amount of bound protein are 10 mg/g with the spectrophotometric method and 6 mg/g with the modified Lowry method. The results reported in this paper should encourage other workers to adopt these basically simple methods and adapt them to proteins immobilized on other carriers.
ANALYSIS
OF
AGAROSE-BOUND
PROTEINS
567
ACKNOWLEDGMENTS Thanks are due to Dr. von Lengerken and Dipl. Chem. Mueller (Zentralinstitut fur Futtermittelpriifung, Lettin) for permission to use the Hitachi spectrofluoriphotometer. The excellent technical assistance of Mrs. G. Hahn, Mrs. C. Winckler and Mrs. I. Zernahle is gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
Axen, R., and Ernback, S. (197 1) Eur. J. Biochem. 18. 351. Hourigan, J. A., and Melrose, G. J. H. (1972) .f. Macromol. Sci. Chem. A6, 761. Havekes, L., Briickmann, F., and Visser, J. (1974) Biochim. Biophys. Acta 334, 272. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L., and Randall, R. J. (195l)J. Biol. Chem. 193, 265. 5. Failla, D., and Santi, D. V. (1973) Anai. Biochem. 52, 363. 6. Hanson, H., Gllsser, D., and Kirschke, H. (1965) Hoppe-Seyler’s Z. Physiol. Chem. 340, 107. 7. Glasser, D., and Kleine, R. (1962) Pharmazie 17, 32. 8. Lasch, J., Iwig, M., and Hanson, H. (1972) Eur. J. Biochem. 27, 431. 9. Laskowski, M., Sr., Kassell, B., Planasky, R. J., and Laskowski. M. Jr., in Handbuch der Physiologisch und Pathologisch-chemischen Analyse Hoppe-Seyler/Thierfelder Bd. VI/C, p. 247. Springer Verlag, BerlinHeidelberg, New York. 1966. 10. Moore, S., Spackman, D. H., and Stein, W. H. (1958) Anal. Chem. 30, 1185; Spackman, D. H., Stein, W. H., and Moore, S. (I 958) Anal. Chem. 30, 1190. 1 I. Damaschun, G., Damaschun, H.. Hanson, H.. Miiller, J. J., and Piirschel, H.-V. (1973) Stud. Biophys. 35, 59. 12. Kretschmer, K., and Hanson, H. (1965) Hoppe-Seyler’s Z. Physiol. Chem. 340, 126. 13. Walsh, K. A., and Neurath, H. (1964) Proc. Nat. Acad. Sci. USA 52, 884. 14. Koelsch, R., Lasch, J., and Hanson, H. (1970) Acta Biol. Med. Ger. 24, 833. 15. Lasch, J. (1974) IV. Diskussionstagung “Biophysik der Eiweipe” der Ceseltschaft fur physikalische und mathematische Biologie der DDR, Potsdam.
BIOCHEMISTRY
Application
66,
556-567 (1975)
of Spectrophotometric the Determination Bound
Methods
to
of Protein
to Agarose
Beads
REGINE KOELSCH, J. LASCH, IRMGARD MARQUARDT, AND H. HANSON Physiologisch-chemisches Institut der Martin-Luther-Universitat, 402 HallelSaale, Hollystrasse l( GDR Received September 20, 1974; accepted January 16, 1975 Quantitation of the amount of leucine aminopeptidase and trypsin covalently attached to Sepharose 6B using the CNBr method was accomplished by five independent methods: protein balance (estimation of the amount of protein not recovered in the final washings), amino acid analysis, modified Lowry method, spectrophotometric and fluorophotometric analysis, the latter two being practicable with the undestroyed gel beads. These methods adapted to the special conditions in particulate enzyme systems were investigated in detail and the results compared. The last three methods were found to be simple, sensitive and highly reproducible.
Attachment of enzymes to insoluble carriers has become a frequently used technique in many laboratories. Especially, the coupling of proteins to polysaccharide supports by the CNBr method is now a very common procedure. The exact determination of the amount of bound protein is highly important for a number of reasons. It is particularly necessary if one has to measure how the enzymic activity has been modified by immobilization (e.g., partial inactivation) or if one has to estimate kinetic parameters of the bound enzyme in comparison with the free enzyme in solution. Accurate protein determinations are also indispensable for the proof of enzyme leaching from the support. If one uses CNBr-activated Sepharose or Sephadex gels, protein evaluations from the nitrogen content of the product are impossible. The most frequently used method is quantitative amino acid analysis after acid hydrolysis of the dried protein-carrier conjugate (l), which seems to have acquired widespread use as standard procedure (2). Another often employed method is to estimate the bound protein by protein “balance”, i.e., to calculate the difference between the amounts of protein added and those found in the supernatant fluid and washings after the coupling reaction. Havekes and co-workers (3) have reported the quantitation of Se556 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
ANALYSIS
OF
AGAROSE-BOUND
PROTEINS
557
pharose-bound glutamate dehydrogenase by a modified Lowry (4) procedure. Spectrophotometric measurements were carried out by Failla and Santi (5) after acid solubilization of the agarose matrix in order to quantitate ligands used in affinity chromatography. To our knowledge, direct sprectrophotometric methods with the intact gel beads have not been used up to now, probably because many workers believe that the insoluble particles interfere with the measurements. By employing special geometric arrangements, we can show, however, that it is possible to measure the absorbance at 280 nm without destruction of the enzyme-carrying particles. It is the aim of our investigation to find out reproducible and accurate methods for protein determinations of matrix-bound enzymes, methods that are easily and quickly handled. For this purpose we compare protein determinations achieved by amino acid analysis after acid hydrolysis with the “balance” method, with a modified Lowry procedure and with a newly developed spectrophotometric method. Moreover, fluorescence intensity measurements using the front-viewing technique are included in the comparison. MATERIALS
AND
METHODS
Mnterials Sepharose 6B was a product of Pharmacia Fine Chemicals, Uppsala, Sweden. Cyanogen bromide was purchased from PH “Dr. Th. Neubauer,” Erfurt, GDR; twice crystallized trypsin from Serva, Heidelberg, GFR. Leucine aminopeptidase once crystallized from bovine eye lenses was routinely prepared in our institute (6). Amberlite IR 120-X8 was purchased from Rohm und Haas, Darmstadt, GFR, and Dowex 5OW-X4, p.a., 50-100 mesh, from Serva, Heidelberg, GFR. Activation
of Sepharose 6B and Coupling
Procedure
Sepharose 6B was thoroughly washed on a glass funnel and brought to twice its original volume by addition of water. This suspension was used for all activations. One milliliter of this mixture had, after drying under reduced pressure over PZ05, an average weight of 18.7 mg. For the activation procedure we took 1O-ml portions of the Sepharose suspension. Activations were carried out at 15°C using a final CNBr concentration of 20-33 mglml. An adjusted pH of 10.5 was attained by automatic addition of 4 N NaOH using a TTT lc Titrator (Radiometer. Copenhagen, Denmark), and the activation was allowed to proceed at this pH for 6 min. After washing the activated gel with water and 0. I M NaHCO, on a G-3 glass filter, the gel was brought to 10 ml with 0.1 M
558
KOELSCH
ET
AL.
NaHC03. The enzyme solution, 2 ml, about IO-20 mg/ml, was added. The mixture was gently shaken overnight. Thereafter it was filtered and the gel washed sequentially with 0.1 M NaHCO,, 1 M KC1 and H20. Then the gel was brought to a final volume of 10 ml by addition of 0.1 M KC1 and stored at 4°C. Protein
Determination
Methods
Method A: Protein balance. The amounts of protein conjugated on the gel particles were estimated from the difference between the initial amounts of protein added and those found in the pooled filtrate and washings. Protein concentrations were measured by a micro Lowry method (4,7). Method B: ModiJied Lowry reaction. The Lowry reaction (4,7) was carried out with 200 ~1 of the enzyme-Sepharose 6B suspension. During the reaction time (140 min) the mixture was gently shaken at room temperature and centrifuged before absorbance readings of the supernatant fraction were taken. Blanks were composed of equal amounts of unactivated Sepharose 6B suspension. Method C: Ultraviolet spectraphotometry. Measurements were carried out in a Unicam SP 800 Spectrophotometer (Pye Unicam Ltd., Cambridge, England) using the constant-temperature cell-housing for second sample-position SP 871 (directly in front of the photomultiplier). The gel was gravity packed in a 2-mm light-path cuvette and unactivated Sepharose 6B placed in the reference beam. The absorbance at 280 nm was chosen for the calculation of protein amounts using the same molar absorption coefficient as for the enzymes in solution (3.26 X lo5 M-‘cm-’ for leucine aminopeptidase (8) and 3.58 X lo4 M-‘cm-’ for trypsin (9)). We define “concentration” operationally as moles of enzyme per liter of packed gel. This concentration can be easily converted to milligrams of bound enzyme per gram of dry-weight support knowing that 1 ml of gravity-packed gel is equivalent to 48 mg dry weight. This relationship was reproducibly obtained from the dry weight of 1 ml of Sepharose 6B suspension and its sedimentation ratio (i.e., total volume: volume of the settled gravity-packed gel beads). Method D: Amino acid analysis. Samples of Sepharose-bound enzymes were washed with distilled water and dried in vacua over phosphorous pentoxide. Acid hydrolysis with 4 N HCl was carried out in heavy-walled tubes under exclusion of oxygen, using weighed amounts of polysaccharide-enzyme conjugates (about 30 mg) or mixtures of dried Sepharose with protein, and 2 ml of acid, with 1.25 &moles norleutine as an internal standard. Before sealing, the tubes were frozen at -72 “C, evacuated, twice warmed in a water bath and again frozen and evacuated. Hydrolysis conditions: Temperature 110 “C, hydrolysis time
ANALYSIS
OF
AGAROSE-BOUND
PROTEINS
559
24 hr. Hydrolyzates were black-colored and cloudy and had to be purified before amino acid analysis by pretreatment on an ion-exchange column. After removal of the hydrochloric acid in a rotary evaporator, the samples were dissolved in 2 ml of 0.02 N HCl, applied either on a column of Amberlite IR 120-X8 and eluted with 25 ml of 1 M ammonia or on a column of Dowex 5OW-X4 and eluted with 25 ml of 10% pyridinelwater (v/v). The dimensions of both columns were 1.0 X 7.0 cm. The residue, taken to dryness three times on the rotary evaporator, could be redissolved in buffer, pH 2.2, conveniently diluted and filtered through hard filter paper. The recovery of amino acids during this purification procedure was proved in separate experiments. Amino acid analyses were performed with an Amino Acid Analyzer Unichrom (Beckman, Munich, GFR) according to the method of Spackman et uf. (10). The protein content was calculated from the estimated amounts of the amino acids aspartic acid, alanine, valine, leucine, and phenylalanine and the separately determined amino acid composition of the enzyme preparations employed for the immobilization. Method E: Fluorescence intensity measurement. Fluorescence emission measurements were done in front-surface viewing geometry with the Hitachi automatic recording fluorescence spectrophotometer, Model 204 (Hitachi, Ltd., Tokyo, Japan). The Sepharose-enzyme conjugates were gravity packed into l-cm2 cuvettes and the sample excited at 295 nm. Blanks consisting of packed Sepharose 6B showed no measurable fluorescence emission at the amplification chosen. Emission spectra of carrier-enzyme conjugates differing in their protein content were recorded and the areas under the recorder tracings measured by a planimeter. RESULTS
We have chosen leucine aminopeptidase (LAP), an oligomeric ( 11) enzyme with a molecular weight of 326,000 (12) and the monomeric trypsin with a molecular weight of 23,800 (13) as model enzymes. The results of protein determinations by methods A, B and C for Sepharose 6B-bound LAP and trypsin are given in Table 1. Method A must be regarded as rather inaccurate, especially in cases where small volumes of washings have to be handled or if the amount of bound protein is small. Nevertheless, one can see from Table 1 that there is, in general, a satisfactory agreement with the results obtained by other methods. Method B seems to be a very simple and accurate method with high reproducibility. For one sample of Sepharose-bound protein, determined three times on different days and after storing at 4°C between the es-
560
KOELSCH TABLE
COMPARISON
OF AMOUNTS
BOUND
PROTEIN
A method Milligrams a
AL.
1 DETERMINED
BY DIFFERENT
B Modified Lowry procedure
“Balance”
Sample
ET
Moles 10’6
Milligrams a
Moles 10’6
METHODS
C Spectrophotometry Milligrams a
Moles 1076
Sepharose 6B-LAP
32.1 49.7 48.2 12.8
1.0 1.5 1.5 0.4
32.8 46.7 41.7 13.4
1.0 1.4 1.3 0.4
29.8 42.4 43.5 14.2
0.9 1.3 1.3 0.4
Sepharose 6B-trypsin
45.5 44.4 83.0 33.7
19.2 18.7 35.0 14.2
38.5 47.1 68.0 39.8
16.2 19.8 28.6 16.6
44.0 47.4 65.0 34.6
18.5 19.9 27.3 14.5
’ Milligrams of enzyme per gram of dried support. * Moles of enzyme per gram of dried support.
timations, we found the following values: 0.59 mg/ml; 0.58 mg/ml; 0.58 mg/ml. At first we attributed the accuracy and reproducibility of this method to quantitative detachment of the protein from the carrier by the prolonged treatment of the suspension at alkaline pH. Appropriate reference experiments revealed, however, that the alkaline medium detached no more than 80%,of the bound protein from the gel beads. A quantitative estimation of bound protein thus necessitates treatment of the enzyme-loaded carrier particles with Folin’s reagent. This led us to the conclusion that the reduced phosphomolybdate complex diffuses freely out of the support. Method C is easy to handle. Gravity packing of the cuvettes is a bit time-consuming (it takes about 2 hr), but this is compensated for by the rapid and convenient measurement. Figures 1 and 2 show the uv spectra of Sepharose-bound leucine aminopeptidase and trypsin in comparison to the free enzymes in solution. The spectra of the bound enzymes are very similar to those of the free ones. It is only in the shorter wavelength region that the spectrum becomes distorted by the background absorption and light scattering of the polysaccharide carrier. Absorption at 280 nm can be read easily. In order to prove the validity of the Lambet-t-Beer law, absorbances were plotted against the amount of protein (LAP) attached to the carrier (Fig. 3). From the resulting straight line we calculate the same absorption coefficient (see Methods) as for the dissolved enzyme. The same holds true with trypsin. This finding
ANALYSIS
OF AGAROSE-BOUND
o.0
561
PROTEINS
0 wavelength
nm
FIG. 1. Ultraviolet spectra of soluble and insolubilized leucine aminopeptidase. I. I.AP dissolved in water; concentration, 1.63 X ~O+M; reference: water. 2, LAP covalently bound to Sepharose 6B. gravity packed: reference: Sepharose 6B: light path. 0.7 cm.
wavelength
nm
FIG. 2. Ultraviolet spectra of soluble and insolubilized trypsin. 1. Trypsin dissolved in IO-"M HCI; concentration, 1.04 X IO-'M: reference: ~O-"M HCI. 1, trypsin covalently attached to Sepharose 6B, gravity packed in a 0.2-cm light path cuvette; reference: unmodified Sepharose 6B.
562
KOELSCH
protein 3 106x
operational
ET
AL.
amount
tmg/g) 6
concentration
9 CM 1
3. Proof of the validity of the Lambert-Beer law for Sepharose-bound leucine aminopeptidase. Sepharose-bound LAP suspensions were pipetted into 0.2-cm light path cuvettes, gravity packed and the absorbance read at 280 nm. Reference: unmodified Sepharose 6B. Amounts of protein were determined by the modified Lowry procedure and by the “balance” method. FIG.
together with the observation that many structural and functional properties of leucine aminopeptidase are not changed upon coupling by the BrCN method (14,15) seem to justify the use of the same molar absorbance coefficient for the free and attached enzyme. It is seen from Table 1 that protein amounts found by this method are in good agreement with those found by Methods A and B. It seems that Method D, which might be believed to yield the most accurate results, has never been evaluated thoroughly and compared with other, particularly destructionless methods. The well-known precision of this method with pure proteins may be lost in the presence of support material. In order to verify that acid hydrolysis in the presence of polysaccharide does not lead to high losses of amino acids, dried Sepharose was mixed with known amounts of leucine aminopeptidase and subjected to the same hydrolysis conditions as the soluble and insoluble enzyme. The results are summarized in Table 2. There is fairly good agreement between the values of the selected five amino acids, pointing to the fact
ANALYSIS
OF
AGAROSE-BOUND
563
PROTEINS
TABLE 2 AMINO ACID ANALYSES: REFERENCE AND CONTROL EXPERIMENTS 1=
2a
30
Amino acid
Ab
B”
Ab
B’
Aspartic acid Alanine Valine Leucine Phenylalanine
0.31 0.35 0.20 0.26 0.12
234 264 1.51 196 91
0.34 0.37 0.20 0.26 0.12
257 219 151 196 91
Ab
B’
Xd
X
X
X
X
X
0.25 0.12
187 91
0 1, Reference: 2.16 mg of leucine aminopeptidase; 2, 2.16 mg of LAP + 29.6 mg of Sepharose 6B, after acid hydrolysis followed by adsorption on a Dowex SOW-X4 column and elution with 10% pyridine; 3, 2.16 mg of LAP + 30.4 mg Sepharose 6B, after acid hydrolysis followed by adsorption on a Dowex 5OW-X4 column. and elution with 1 M ammonia. b Micromoles amino acid per milliliter of hydrolyzate. c Number of residues. d x, Overlapping peaks, estimation not possible.
that, at least for these amino acids, interference by the added polysaccharide material is negligible. In experiments where the hydrolyzed sample was eluted from the prewash column (either Amberlite or Dowex) by 1 M ammonia it was consistently found that the subsequent chromatography does not result in sharp peaks of aspartic acid, alanine and valine. These peak distortions are probably caused by undesired secondary hydrolysis products. With the emergence of the second buffer according to the standard procedure, (10) leucine and phenylalanine, however, emerged from the column as the usual sharp peaks. In these cases, the calculation of the amount of protein was based only on leucine and phenylalanine. These difficulties could be avoided in later experiments by employing a Dowex SOW-X4 prewash column and eluting with 10% pyridine. The results of amino acid analysis are summarized in Table 3 along with the values obtained by other methods. The finding that the amounts of bound protein as determined by amino acid analysis are consistently lower than those estimated by other methods may be explained by appreciable side-reactions of the derivatized carrier during acid hydrolysis. Interestingly, the hydrolysis side-reactions of derivatized and underivatized Sepharose seem to be quite different under otherwise identical conditions of hydrolysis as already evident by a difference in the color of the hydrolyzates. Method E utilizes the linear proportionality between fluorescence signal and fluorochrome concentration that holds for sufficiently small concentrations for which the usual exponential relation between measur-
Pretreatment
Sample
OF BOUND
value from “balance” method from Lowry method from spectrophotometry
PROTEIN
TABLE
3
26.81 28.79 17.27 21.11 11.52
A”
1.15 1.5 1.3 1.3
1.15 1.09 1.14 1.08 1.27
B* 8.52 8.52 4.26 7.1 2.84
A”
3.3 3.9 4.0 4.4
3.6 3.2 2.8 3.6 3.1
Bb
Dowex 50-X4, pyridine
13.68 16.72 7.60 9.12 4.56
A”’
Sepharose 6B-leucine
5.4 6.0 4.6
5.9 6.3 5.0 4.7 5.0
B”
5.9 9.0 6.5
X
5.7 6.1
X
x
X
B”
Amberlite,
11.15 5.58
XC X
A”
aminopeptidase
17.36
X
X
X X
A”
1.9 2.6 2.0
1.9
X
X
X X
Bb
“BALANCE”
ammonia
AS DETERMINED BY AMINO ACID ANALYSIS AFTER ACID HYDROLYSIS. THE MODIFIED LOWRY REACTION, AND THE SPECTROPHOTOMETRIC METHOD
UA, Micromoles of amino acid per gram of dried support. * B, Moles of enzyme per gram of dried support. COverlapping peaks, estimation not possible,
Mean Value Value Value
Aspartic acid Alanine Valine Leucine Phenylalanine
-
AMOUNT
THE
36.00 24.32 24.32 25.30 5.84
Afl
2.0 3.5 2.9 2.7
1.89 2.03 2.03 2.11 1.95
Bb
Dowex 50-X4, pyridine
Sepharose 6B-trypsin
METHOD,
.F
2
i
6 F
ANALYSIS
OF AGAROSE-BOUND
565
PROTEINS
1
J
3 lo6
x operational
6 concentration(M
9 1
FIG. 4. Measured areas under the fluorescence intensity spectra plotted against amounts of protein estimated by other methods. 0. Protein determined by spectrophotometry: Cl. protein determined by “balance” method. For conditions, see Methods.
able fluorescence intensity F and concentration, F = F,+ (1 - P-“.:~~~~~~“‘), simplifies to F = 2.303 F&cd, where F, = excitation energy, 4 = relative quantum efficiency, E = molar absorption coefficient (M-‘cm-‘), c= molar concentration, and d = thickness (cm). Figure 4 demonstrates that the linear correlation between fluorescence intensity and amount of enzyme bound can be readily used to quantitate insolubilized proteins. The areas under the emission spectra were measured in connection with the evaluation of apparent quantum yields. Intensity measurements at the wavelength of the maximum would yield the same linear correlation. The front-surface viewing technique allows even dense enzyme-carrier suspensions to be determined. DISCUSSION
Failla and Santi (5) described a number of methods for solubilizing derivatized agarose beads permitting quantitative spectrophotometric analysis of covalently attached ligands. Although this method seems to work satisfactorily when the solubilization is effected by 50% acetic
566
KOELSCH
ET
AL.
acid, we found that this procedure may run aground in a number of instances. First, at the usual degree of Sepharose activation by cyanogen bromide, generally not less than 20 mg of BrCN per ml, Sepharose-protein conjugates can not be dissolved completely by treatment with 50% acetic acid at 70°C in contrast to underivatized Sepharose 6B which gives a clear solution after the same treatment. This is readily attributed to cross-linking during the activation step. Second, the spectra of the supernatant portions of acetic acid-treated Sepharose-enzyme conjugates showed distortions probably due to light-absorbing decomposition products of the matrix or, more likely, of the introduced functional groups. Warming the gel beads in acidic or alkaline media confirmed the observations reported by the authors cited above (5) that solubilization in 0.1 M HCI (15 min, 100°C) and I M NaOH produced byproducts that prohibited a quantitative analysis of the spectra. It follows that only appropriately modified and solubilized Sepharose (a nonabsorbing compound like glycine may be coupled) is the correct substance to be used in the reference cuvette in order to blank out uncontrollable background absorbance. Third, the spectra of unmodified Sepharose set lower limits to the spectrophotometric measurement of the concentration of bound ligands. The concentrations encountered in experiments with immobilized enzymes are far below these limits. In an attempt to overcome these difficulties we tried successfully to adapt spectrophotometric and lluorophotometric assays to undestroyed Sepharose beads. One principal problem that arises is how to calibrate the different methods. We chose for calibration and comparison the amino acid analysis and the modified Lowry method, the latter giving highly reproducible results. In addition, the agreement between the values obtained by different and independent methods confirms the usefulness of the investigated methods. The finding that the amino acid analysis consistently affords values of the amount of bound protein that are too low seems to cast doubt on the usefulness of this method in the presence of high amounts of polysaccharide carrier. That this is not true with immobilized oligopeptides (R. Yost and A. Jarron, personal communication) may be explained by the less extensive side-reactions in the hydrolysis mixture. The lower limits for the determination of the amount of bound protein are 10 mg/g with the spectrophotometric method and 6 mg/g with the modified Lowry method. The results reported in this paper should encourage other workers to adopt these basically simple methods and adapt them to proteins immobilized on other carriers.
ANALYSIS
OF
AGAROSE-BOUND
PROTEINS
567
ACKNOWLEDGMENTS Thanks are due to Dr. von Lengerken and Dipl. Chem. Mueller (Zentralinstitut fur Futtermittelpriifung, Lettin) for permission to use the Hitachi spectrofluoriphotometer. The excellent technical assistance of Mrs. G. Hahn, Mrs. C. Winckler and Mrs. I. Zernahle is gratefully acknowledged.
REFERENCES 1. 2. 3. 4.
Axen, R., and Ernback, S. (197 1) Eur. J. Biochem. 18. 351. Hourigan, J. A., and Melrose, G. J. H. (1972) .f. Macromol. Sci. Chem. A6, 761. Havekes, L., Briickmann, F., and Visser, J. (1974) Biochim. Biophys. Acta 334, 272. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L., and Randall, R. J. (195l)J. Biol. Chem. 193, 265. 5. Failla, D., and Santi, D. V. (1973) Anai. Biochem. 52, 363. 6. Hanson, H., Gllsser, D., and Kirschke, H. (1965) Hoppe-Seyler’s Z. Physiol. Chem. 340, 107. 7. Glasser, D., and Kleine, R. (1962) Pharmazie 17, 32. 8. Lasch, J., Iwig, M., and Hanson, H. (1972) Eur. J. Biochem. 27, 431. 9. Laskowski, M., Sr., Kassell, B., Planasky, R. J., and Laskowski. M. Jr., in Handbuch der Physiologisch und Pathologisch-chemischen Analyse Hoppe-Seyler/Thierfelder Bd. VI/C, p. 247. Springer Verlag, BerlinHeidelberg, New York. 1966. 10. Moore, S., Spackman, D. H., and Stein, W. H. (1958) Anal. Chem. 30, 1185; Spackman, D. H., Stein, W. H., and Moore, S. (I 958) Anal. Chem. 30, 1190. 1 I. Damaschun, G., Damaschun, H.. Hanson, H.. Miiller, J. J., and Piirschel, H.-V. (1973) Stud. Biophys. 35, 59. 12. Kretschmer, K., and Hanson, H. (1965) Hoppe-Seyler’s Z. Physiol. Chem. 340, 126. 13. Walsh, K. A., and Neurath, H. (1964) Proc. Nat. Acad. Sci. USA 52, 884. 14. Koelsch, R., Lasch, J., and Hanson, H. (1970) Acta Biol. Med. Ger. 24, 833. 15. Lasch, J. (1974) IV. Diskussionstagung “Biophysik der Eiweipe” der Ceseltschaft fur physikalische und mathematische Biologie der DDR, Potsdam.