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[47] Purification and assays of bacterial gelatinases
602
E N Z Y M E A N D T O X I N ASSAYS
147]
many), and Fluka; both peptides can be made as a 5 m M stock solution in dimethyl sulfoxide and are used at final concentration of 0.25 mM. I M HCI Ethyl acetate, to extract DNP-PLGIAGR Ethyl acetate/n-butanol (1:0.15, v/v), to extract DNP-PQGIAGQR Na2SO4 Procedure. The peptide, assay buffer, and enzyme in a final volume of 0.25 ml are incubated at 37° for up to l hr. The reaction is stopped by the addition of 1 ml of 1 M HC1, and the peptide product is extracted by 1 ml of the appropriate organic solvent, vigorous vortexing, and centrifugation at 500 g to separate the two layers. The A365of the upper organic layer is measured and the amount of peptide product determined using a standard curve.
[47] P u r i f i c a t i o n a n d A s s a y s o f B a c t e r i a l G e l a t i n a s e s
By JOHN D. GRUBB Introduction Bacterial gelatinases may have a significant role in pathogenesis by degrading Type IV collagen in basement membrane and exposing underlying tissue. They may also degrade partially denatured collagen fragments generated either by interstitial collagenase during normal connective tissue metabolism or by bacterial collagenase. The purpose of this chapter is to give a brief description of the chromatographic matrices which may be useful for the selective purification of gelatinases and enzymatic assays using gelatin as substrate. Purification of Gelatinases by Affinity Chromatography Affinity chromatography exploits to varying degrees the selective but reversible interaction of a protein with a ligand which has been immobilized on a chromatographic support. The ligands described below can be divided into two groups defined by their interaction with gelatinases. In the first, the binding of gelatinases to gelatin or other extracellular matrix molecules is most likely determined by three-dimensional structural features present in both proteins. In the second, the ligands are small molecules which bind to specific amino acids or metal cofactors. The principles METHODS IN ENZYMOLOGY, VOL. 235
Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.
[47]
PURIFICATION AND ASSAYS OF GELATINASES
603
and the wide variety of practical applications of affinity chromatography have been described in detail previously.~-3 Although many of the ligands described below are available already coupled to a support, a previous volume in this series has described the methods available to immobilize ligands through different functional groups)
Gelatin-Agarose Gelatin-agarose is available commercially, or it can be prepared by coupling gelatin to agarose preactivated with cyanogen bromide. 4 The loading capacity of this material will vary depending not only on the amount of immobilized gelatin but also on the number and type of interactions between the extract proteins and gelatin. Bound proteins ar e eluted from the gelatin using 1 M NaCI, 1 M arginine, t or 7.5% (v/v) dimethyl sulfoxide. 5 Prior to assaying the eluted fractions for enzymatic activity, it may be necessary to remove the eluting agent, in particular the latter two, by dialysis.
Protease Inhibitor-Agarose Matrices Chromatography on inhibitor-agarose matrices relies on the specific interaction of protease inhibitors with individual amino acid residues in the gelatinase active site. Therefore, if the gelatinase is inhibited by a reversible protease inhibitor, binding the gelatinase to the immobilized inhibitor may result in a significant enhancement of purity. Many of these matrices will not be available commercially and, therefore, must be prepared. Preactivated matrices are available or can be prepared as described previously) Because most of the ligands will be small molecules, the accessibility and orientation may be very important. Attachment of the ligand to the support through a spacer molecule may make the ligand more accessible for binding. It may be necessary to bind the ligand through different functionalities in order to determine the optimum orientation of the ligand. Amino acids or peptides which act as reversible inhibitors might also be useful as ligands. As the interaction between the protein and the ligands will most likely be 1 : 1, the capacity will be primarily dependent on the i S. Osgrove, this series, Vol. 182, p. 357. 2 S. Osgrove and S. Weiss, this series, Vol. 182, p. 371. 3 T. M. Phillips, in "Chromatography" (E. Heftmann, ed.), p. A309 Elsevier, New York, 1992. 4 S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). 5 j. G. Lyons, B. Birkedal-Hansen, W. G. I. Moore, R. L. O'Grady, and H. BirkedalHansen, Biochemistry 30, 1449 (1991).
604
ENZYME AND TOXIN ASSAYS
[47]
concentration of bound ligand and steric interference between bound proteins. Elution may be accomplished in three ways: (1) high salt, (2) a change in pH, and (3) competition by free ligand for the ligand binding site. 3
Thiol-Disulfide Covalent Chromatography Thiol-disulfide covalent chromatography relies on the presence of cysteine residues in protein, in particular, one reactive thiol per molecule, as found in cysteine proteases. The technique has been described previously so only a brief description f o l l o w s . 6'7 Typically, glutathione-agarose is used, in which the glutathione is coupled to the agarose via the amino group so that the thiol group is available for binding. The thiol groups of glutathione-agarose are reacted with 2,2'-dipyridyl disulfide to produce a polymer containing glutathione-2-pyridyl disulfide residues with the release of 2-thiopyridone. Attachment of the thiol-containing protein to the column results in the release of 2-thiopyridone, which is detectable spectrophotometrically a t A343. At pH 8, most thiol-containing molecules will react readily. Further selectivity for thiol groups with very low PKa values, such as found in many cysteine proteases, may be achieved in acidic media (e.g., pH 4). In both cases, thiol-containing proteins are then eluted with a gradient of cysteine.
Immobilized Metal Affinity Chromatography The immobilized metal affinity chromatography (IMAC) technique is particularly useful in the purification of proteases known to bind a divalent metal ion, for example, Zn 2+ or Ca 2+. A collagenase 8 and a gelatinase, 9 both of which are zinc-dependent proteases, have been purified using the resin. In addition, the resin was used in the purification of calcium-binding proteins. ~° The IMAC technique was developed in the mid-1970s by Porath, and the principles and procedure have been described previously. 2
6 K. Brocldehurst, J. Carlsson, M. P. J. Kierstan, and E. M. Crook, this series, Vol. 34, p. 531. 7 K. Brocklehurst, J. Carlsson, and M. P. J. Kierstan, Top. Enzyme Ferment. Biotechnol. 10, 146 (1985). s T. E. Cawston and J. A. Tyler, Biochem. J. 183, 647 (1979). 9 L. Smilenov, E. Forsberg, I. Zeligman, M. Sparrman, and S. Johansson, FEBS Lett. 302, 227 (1992). l0 j. A. Campbell, J. D. Biggart, and R. J. Elliott, Biochem. Soc. Trans. 19, 387S (1991).
[47]
PURIFICATION AND ASSAYS OF GELATINASES
605
Assays of Gelatinase Gelatin Preparation Gelatin for the assays described below can be purchased, although commercial preparations can have a large size range of polypeptide chains. A homogeneous gelatin preparation can be obtained by heating acid- or pepsin-solubilized collagen at 56° for 15 min. Type IV collagen may be substituted for gelatin to determine if the gelatinase degrades basement collagen.
Electrophoresis Assay Gelatin, at 50 /~g/ml, is incubated with the enzyme fraction for an appropriate amount of time. The reactions should be stopped either by an inhibitor of the enzyme or by acidification since some gelatinases are capable of renaturing following boiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The degradation of gelatin is visualized by subjecting the reactions to 10% SDS-PAGE followed by silver staining. Detection may be visualized by Coomassie staining if the gelatin concentration is increased. The greatest advantage to this assay is its simplicity, whereas its usefulness is limited by the inability to quantitate degradation.
Ninhydrin Detection of a-Amino Groups Successive cleavages of the gelatin polypeptide chains result in an increasing number of new a-amino groups, which are detectable colorimetrically using ninhydrin. Among the several variations of this procedure, 11-13 the one described here 13is the simplest to perform and does not have the unpleasant odor associated with the use of organic solvents in other procedures, while retaining high sensitivity.
Materials 0.5 M Sodium citrate (pH 5.5) 1% Ninhydrin in 0.5 M citrate Glycerol Procedure. For each sample, mix together 0.5 ml of 1% ninhydrin, 1.2 ml of glycerol, and 0.2 ml of 0.5 M citrate. Dispense 1.9 ml/tube, add II H. Rosen, Arch. Biochem. Biophys. 67, 10 (1957). 12 j. V. Singh, S. K. Khanna, and G. B. Singh, Anal. Biochem. 85, 581 (1978). 13 y . p. Lee and T. Takahashi, Anal. Biochem. 14, 71 (1966).
606
ENZYME AND TOXIN ASSAYS
[48]
sample in 0.1 ml, and heat in a boiling water bath for 12 min. Cool in a water bath at room temperature. Shake each tube and read A570within I hr. Assays Using Labeled Gelatin Gelatin can be radiolabeled with [3H]acetic anhydride or [3H]formaldehyde, as done for collagen.~4,~5Alternatively, it can be fluorescently labeled with fluorescein isothiocyanate (FITC).16 Incubate enzyme with radiolabeled gelatin or FITC-gelatin in a volume of 50 /~1 for an appropriate amount of time at 37°. The reactions are stopped by adding 100/~1 of 10 mg/ml unlabeled gelatin and 50/.d of 50% trichloroacetic acid (TCA). Let the reactions stand at 4° for 30 min and then pellet the protein precipitate by centrifugation in a microcentrifuge. In the case of radiolabeled gelatin, the supernatant is counted to determine the counts per minute (cpm). The FITC-labeled peptides in the supernatant are detected by adding 100 /~1 to 1.0 ml of 0.5 M Tris (pH 8.75) and measuring the fluorescence using excitation at 490 nm and monitoring emission at 525 nm. Rather than incubating the labeled gelatin in solution, it can also be covalently attached to the preactivated surface of a 96-well plate (Costar, Cambridge, MA), with release of label into the supernatant being measured. 14 H. Birkedal-Hansen, this series, Vol. 144, p. 140. 15 K. A. Mookhtiar, S. K. Mallya, and H. E. Van Wart, Anal. Biochem. 158, 322 (1986). 16 U. Tisljar and H . - W . Denker, Anal. Biochem. 152, 39 (1986).
[48] A s s a y s for H y a l u r o n i d a s e A c t i v i t y
By WAYNE L. HYNES and JOSEPH J. FERRETTI Introduction Hyaluronidases are a group of enzymes [hyaluronate 4-glycanohydrolase (EC 3.2.1.35, hyaluronoglucosamidase), hyaluronate 3-glycanohydrolase (EC 3.2.1.36, hyaluronoglucuronidase), and hyaluronate lyase (EC 4.2.2.1)] that catalyze the breakdown of hyaluronic acid, a mucopeptide composed of alternating N-acetylglucosamine and glucuronic acid residues. Hyaluronidases are produced by a variety of pathogenic organisms including group A and C streptococci, pneumococci, staphylococci, and clostridia. The enzymes are also found in leeches, snake and insect venoms, and malignant tissues. In the pathogens, hyaluronidases are freMETHODS IN ENZYMOLOGY, VOL. 235
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
E N Z Y M E A N D T O X I N ASSAYS
147]
many), and Fluka; both peptides can be made as a 5 m M stock solution in dimethyl sulfoxide and are used at final concentration of 0.25 mM. I M HCI Ethyl acetate, to extract DNP-PLGIAGR Ethyl acetate/n-butanol (1:0.15, v/v), to extract DNP-PQGIAGQR Na2SO4 Procedure. The peptide, assay buffer, and enzyme in a final volume of 0.25 ml are incubated at 37° for up to l hr. The reaction is stopped by the addition of 1 ml of 1 M HC1, and the peptide product is extracted by 1 ml of the appropriate organic solvent, vigorous vortexing, and centrifugation at 500 g to separate the two layers. The A365of the upper organic layer is measured and the amount of peptide product determined using a standard curve.
[47] P u r i f i c a t i o n a n d A s s a y s o f B a c t e r i a l G e l a t i n a s e s
By JOHN D. GRUBB Introduction Bacterial gelatinases may have a significant role in pathogenesis by degrading Type IV collagen in basement membrane and exposing underlying tissue. They may also degrade partially denatured collagen fragments generated either by interstitial collagenase during normal connective tissue metabolism or by bacterial collagenase. The purpose of this chapter is to give a brief description of the chromatographic matrices which may be useful for the selective purification of gelatinases and enzymatic assays using gelatin as substrate. Purification of Gelatinases by Affinity Chromatography Affinity chromatography exploits to varying degrees the selective but reversible interaction of a protein with a ligand which has been immobilized on a chromatographic support. The ligands described below can be divided into two groups defined by their interaction with gelatinases. In the first, the binding of gelatinases to gelatin or other extracellular matrix molecules is most likely determined by three-dimensional structural features present in both proteins. In the second, the ligands are small molecules which bind to specific amino acids or metal cofactors. The principles METHODS IN ENZYMOLOGY, VOL. 235
Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.
[47]
PURIFICATION AND ASSAYS OF GELATINASES
603
and the wide variety of practical applications of affinity chromatography have been described in detail previously.~-3 Although many of the ligands described below are available already coupled to a support, a previous volume in this series has described the methods available to immobilize ligands through different functional groups)
Gelatin-Agarose Gelatin-agarose is available commercially, or it can be prepared by coupling gelatin to agarose preactivated with cyanogen bromide. 4 The loading capacity of this material will vary depending not only on the amount of immobilized gelatin but also on the number and type of interactions between the extract proteins and gelatin. Bound proteins ar e eluted from the gelatin using 1 M NaCI, 1 M arginine, t or 7.5% (v/v) dimethyl sulfoxide. 5 Prior to assaying the eluted fractions for enzymatic activity, it may be necessary to remove the eluting agent, in particular the latter two, by dialysis.
Protease Inhibitor-Agarose Matrices Chromatography on inhibitor-agarose matrices relies on the specific interaction of protease inhibitors with individual amino acid residues in the gelatinase active site. Therefore, if the gelatinase is inhibited by a reversible protease inhibitor, binding the gelatinase to the immobilized inhibitor may result in a significant enhancement of purity. Many of these matrices will not be available commercially and, therefore, must be prepared. Preactivated matrices are available or can be prepared as described previously) Because most of the ligands will be small molecules, the accessibility and orientation may be very important. Attachment of the ligand to the support through a spacer molecule may make the ligand more accessible for binding. It may be necessary to bind the ligand through different functionalities in order to determine the optimum orientation of the ligand. Amino acids or peptides which act as reversible inhibitors might also be useful as ligands. As the interaction between the protein and the ligands will most likely be 1 : 1, the capacity will be primarily dependent on the i S. Osgrove, this series, Vol. 182, p. 357. 2 S. Osgrove and S. Weiss, this series, Vol. 182, p. 371. 3 T. M. Phillips, in "Chromatography" (E. Heftmann, ed.), p. A309 Elsevier, New York, 1992. 4 S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). 5 j. G. Lyons, B. Birkedal-Hansen, W. G. I. Moore, R. L. O'Grady, and H. BirkedalHansen, Biochemistry 30, 1449 (1991).
604
ENZYME AND TOXIN ASSAYS
[47]
concentration of bound ligand and steric interference between bound proteins. Elution may be accomplished in three ways: (1) high salt, (2) a change in pH, and (3) competition by free ligand for the ligand binding site. 3
Thiol-Disulfide Covalent Chromatography Thiol-disulfide covalent chromatography relies on the presence of cysteine residues in protein, in particular, one reactive thiol per molecule, as found in cysteine proteases. The technique has been described previously so only a brief description f o l l o w s . 6'7 Typically, glutathione-agarose is used, in which the glutathione is coupled to the agarose via the amino group so that the thiol group is available for binding. The thiol groups of glutathione-agarose are reacted with 2,2'-dipyridyl disulfide to produce a polymer containing glutathione-2-pyridyl disulfide residues with the release of 2-thiopyridone. Attachment of the thiol-containing protein to the column results in the release of 2-thiopyridone, which is detectable spectrophotometrically a t A343. At pH 8, most thiol-containing molecules will react readily. Further selectivity for thiol groups with very low PKa values, such as found in many cysteine proteases, may be achieved in acidic media (e.g., pH 4). In both cases, thiol-containing proteins are then eluted with a gradient of cysteine.
Immobilized Metal Affinity Chromatography The immobilized metal affinity chromatography (IMAC) technique is particularly useful in the purification of proteases known to bind a divalent metal ion, for example, Zn 2+ or Ca 2+. A collagenase 8 and a gelatinase, 9 both of which are zinc-dependent proteases, have been purified using the resin. In addition, the resin was used in the purification of calcium-binding proteins. ~° The IMAC technique was developed in the mid-1970s by Porath, and the principles and procedure have been described previously. 2
6 K. Brocldehurst, J. Carlsson, M. P. J. Kierstan, and E. M. Crook, this series, Vol. 34, p. 531. 7 K. Brocklehurst, J. Carlsson, and M. P. J. Kierstan, Top. Enzyme Ferment. Biotechnol. 10, 146 (1985). s T. E. Cawston and J. A. Tyler, Biochem. J. 183, 647 (1979). 9 L. Smilenov, E. Forsberg, I. Zeligman, M. Sparrman, and S. Johansson, FEBS Lett. 302, 227 (1992). l0 j. A. Campbell, J. D. Biggart, and R. J. Elliott, Biochem. Soc. Trans. 19, 387S (1991).
[47]
PURIFICATION AND ASSAYS OF GELATINASES
605
Assays of Gelatinase Gelatin Preparation Gelatin for the assays described below can be purchased, although commercial preparations can have a large size range of polypeptide chains. A homogeneous gelatin preparation can be obtained by heating acid- or pepsin-solubilized collagen at 56° for 15 min. Type IV collagen may be substituted for gelatin to determine if the gelatinase degrades basement collagen.
Electrophoresis Assay Gelatin, at 50 /~g/ml, is incubated with the enzyme fraction for an appropriate amount of time. The reactions should be stopped either by an inhibitor of the enzyme or by acidification since some gelatinases are capable of renaturing following boiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The degradation of gelatin is visualized by subjecting the reactions to 10% SDS-PAGE followed by silver staining. Detection may be visualized by Coomassie staining if the gelatin concentration is increased. The greatest advantage to this assay is its simplicity, whereas its usefulness is limited by the inability to quantitate degradation.
Ninhydrin Detection of a-Amino Groups Successive cleavages of the gelatin polypeptide chains result in an increasing number of new a-amino groups, which are detectable colorimetrically using ninhydrin. Among the several variations of this procedure, 11-13 the one described here 13is the simplest to perform and does not have the unpleasant odor associated with the use of organic solvents in other procedures, while retaining high sensitivity.
Materials 0.5 M Sodium citrate (pH 5.5) 1% Ninhydrin in 0.5 M citrate Glycerol Procedure. For each sample, mix together 0.5 ml of 1% ninhydrin, 1.2 ml of glycerol, and 0.2 ml of 0.5 M citrate. Dispense 1.9 ml/tube, add II H. Rosen, Arch. Biochem. Biophys. 67, 10 (1957). 12 j. V. Singh, S. K. Khanna, and G. B. Singh, Anal. Biochem. 85, 581 (1978). 13 y . p. Lee and T. Takahashi, Anal. Biochem. 14, 71 (1966).
606
ENZYME AND TOXIN ASSAYS
[48]
sample in 0.1 ml, and heat in a boiling water bath for 12 min. Cool in a water bath at room temperature. Shake each tube and read A570within I hr. Assays Using Labeled Gelatin Gelatin can be radiolabeled with [3H]acetic anhydride or [3H]formaldehyde, as done for collagen.~4,~5Alternatively, it can be fluorescently labeled with fluorescein isothiocyanate (FITC).16 Incubate enzyme with radiolabeled gelatin or FITC-gelatin in a volume of 50 /~1 for an appropriate amount of time at 37°. The reactions are stopped by adding 100/~1 of 10 mg/ml unlabeled gelatin and 50/.d of 50% trichloroacetic acid (TCA). Let the reactions stand at 4° for 30 min and then pellet the protein precipitate by centrifugation in a microcentrifuge. In the case of radiolabeled gelatin, the supernatant is counted to determine the counts per minute (cpm). The FITC-labeled peptides in the supernatant are detected by adding 100 /~1 to 1.0 ml of 0.5 M Tris (pH 8.75) and measuring the fluorescence using excitation at 490 nm and monitoring emission at 525 nm. Rather than incubating the labeled gelatin in solution, it can also be covalently attached to the preactivated surface of a 96-well plate (Costar, Cambridge, MA), with release of label into the supernatant being measured. 14 H. Birkedal-Hansen, this series, Vol. 144, p. 140. 15 K. A. Mookhtiar, S. K. Mallya, and H. E. Van Wart, Anal. Biochem. 158, 322 (1986). 16 U. Tisljar and H . - W . Denker, Anal. Biochem. 152, 39 (1986).
[48] A s s a y s for H y a l u r o n i d a s e A c t i v i t y
By WAYNE L. HYNES and JOSEPH J. FERRETTI Introduction Hyaluronidases are a group of enzymes [hyaluronate 4-glycanohydrolase (EC 3.2.1.35, hyaluronoglucosamidase), hyaluronate 3-glycanohydrolase (EC 3.2.1.36, hyaluronoglucuronidase), and hyaluronate lyase (EC 4.2.2.1)] that catalyze the breakdown of hyaluronic acid, a mucopeptide composed of alternating N-acetylglucosamine and glucuronic acid residues. Hyaluronidases are produced by a variety of pathogenic organisms including group A and C streptococci, pneumococci, staphylococci, and clostridia. The enzymes are also found in leeches, snake and insect venoms, and malignant tissues. In the pathogens, hyaluronidases are freMETHODS IN ENZYMOLOGY, VOL. 235
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.