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Calibrating Gelatin Zymograms with Human Gelatinase Standards
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
236, 353–356 (1996)
0179
Calibrating Gelatin Zymograms with Human Gelatinase Standards Gregory S. Makowski* and Melinda L. Ramsby† *Department of Laboratory Medicine, †School of Medicine, MC-2235, University of Connecticut School of Medicine, Farmington, Connecticut 06030
Received December 4, 1995
We describe the use of gelatinase standards from human capillary whole blood for calibrating gelatin zymograms. Capillary blood was obtained by fingerstick puncture and prepared in nonreducing Laemmli SDS – PAGE sample buffer without heating. Gelatin zymography revealed that whole blood (0.25 – 1 ml) contained substantial fibroblast-derived (72kDa) and neutrophil-derived (92-, 130-, and 225-kDa) gelatinases which could be used for calibration purposes. Calibration was linear under different electrophoretic conditions using 6.5– 10% polyacrylamide gels containing 1 –2 mg/ml gelatin (correlation range, r Å 0.990 – 0.998; perfect correlation, r Å 1.000). The use of the standards for characterizing gelatinases from arthritic joint fluid is demonstrated. The standards are easily obtained and well characterized, and should facilitate interlaboratory comparison and standardization. q 1996 Academic Press, Inc.
Matrix metalloproteinases (MMP)1 are a diverse group of collagen-degrading enzymes implicated as playing a pivotal, although poorly defined, role in pathologic states characterized by aberrant inflammatory or invasive processes. Instrumental for elucidating the role of MMP in disease is the technique of gelatin zymography, an SDS–PAGE-based method performed with copolymerized gelatin (1). Inherent to gelatin zymography is the capacity to simultaneously detect both activated and latent forms of MMP while concomitantly resolving MMP of similar as well as diverse molecular weight (Mr). Gelatin zymography thus enables investigation of the full spectrum of MMP subclasses and iso1 Abbreviations used: MMP, matrix metalloproteinases; PMN, polymorphonuclear leukocytes; ACD, acid–citrate–dextrose; OA, osteoarthritis; RA, rheumatoid arthritis; SA, septic arthritis.
forms, the relative proportions of which appear to correlate with disease state and severity (2, 3). Despite its numerous applications, the utility of gelatin zymography is limited by poor interlaboratory comparison due to lack of a readily available MMP Mr reference standard. Thus, investigators have used no standards, standards of unclear origin, or ‘‘in-house’’ MMP purified by labor-intensive chromatographic methods (3–6). Toward the goal of defining a readily available MMP Mr standard, we investigated the feasibility of using a fingerstick blood sample as a reference material. Such samples are easily obtained and contain MMP of white blood cell and fibroblastic origin, the Mr of which have been well characterized (7, 8). MATERIALS AND METHODS
Materials Acrylamide and N,N*-methylene-bis-acrylamide were from Boehringer Mannheim Corp. (Indianapolis, IN). Vacutainer collection tubes and Flow Lancet were from Becton Dickinson Corp. (Rutherford, NJ). Ficoll–Hypaque was from LKB-Pharmacia (Piscataway, NJ). Unless specified, all other reagents were from Sigma Chemical Co. (St. Louis, MO). Methods Preparation of samples. A drop of human capillary blood (15–30 ml) was obtained by fingerstick puncture and placed in a tared polypropylene tube. The sample was weighed and 20 vol nonreducing Laemmli sample buffer (1, 9) was immediately added. The sample was then vortex mixed (30 s) and aliquots stored (0307C, stability 3–6 months). Polymorphonuclear leukocytes (PMN) were obtained from acid–citrate–dextrose (ACD) anticoagulated whole blood following dextran sedimentation, Ficoll–Hypaque centrifugation, and 353
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6m15$$9503
04-03-96 12:27:44
abas
AP-Anal Bio
354
MAKOWSKI AND RAMSBY
FIG. 1. Gelatin zymography of capillary whole blood gelatinases. Zymograms were incubated in the presence (/Ca) and absence (0Ca) of calcium, and calcium plus a zinc chelator (/Ca/0Zn). Neutrophil (N) and plasma (P) gelatinases were used for comparison. Samples (15 ml) were analyzed on 7.5% gels containing 1 mg/ml gelatin (1). Molecular weights (kDa) are indicated.
hypotonic lysis (7). PMN purity routinely averaged 95– 98%. Gelatinases were released from PMN (3000/ml) by freeze–thaw in 1 M NaCl (7) and prepared as described above. Plasma was immediately obtained following centrifugation of sodium citrate anticoagulated whole blood. Synovial fluid was collected in the presence of FIG. 3. Effect of polyacrylamide concentration (6.5–10% gel) on gelatinase standard resolution under (A) Laemmli and (B) modified pH conditions. Standards (20 ml) were analyzed on zymograms containing 1.5 mg/ml gelatin. Molecular weights (kDa) are indicated.
EDTA, cell counts performed, and cleared by centrifugation (10,000g, 20 min). Plasma and synovial fluid were prepared in nonreducing sample buffer as described above. Gelatin zymography. Gelatin zymography (1) was performed on 6.5–10% polyacrylamide gels (11 cm height, 0.75 mm thickness) containing 1–2 mg/ml gelatin under Laemmli (9) or modified pH (resolving gel pH adjusted to 9.2) conditions (10). Following electrophoresis (20 mA, 257C), gels were washed twice in 200 ml 2.5% Triton X-100 (30 min each) and incubated in 100 ml 50 mM Tris–HCl, 5 mM CaCl2 , pH 7.6 (18–20 h, 377C). For inhibition studies, zymograms were incubated in the absence of calcium or in the presence of calcium plus 5 mM 1,10-phenanthroline. Gels were stained with 0.2% Coomassie brilliant blue R-250 (50% methanol, 10% acetic acid) and destained appropriately (40% methanol, 10% acetic acid). Gelatinase activity was evident as cleared (unstained) regions. RESULTS AND DISCUSSION FIG. 2. Effect of gelatin concentration (1, 1.5, and 2 mg/ml) on gelatinase standard resolution under (A) Laemmli and (B) modified pH conditions. Lanes: 1, 5; 2, 10; 3, 15; and 4, 20 ml loaded/lane (7.5% gels used). Molecular weights (kDa) are indicated.
/ 6m15$$9503
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Capillary Gelatinase Characterization Zymography of capillary blood demonstrated four gelatinases that migrated identically to those present
AP-Anal Bio
CALIBRATION OF GELATIN ZYMOGRAMS
355
uated (Figs. 3A and 3B). Although good activity was apparent over the percentage gel range, some obscuring was observed for the largest gelatinase standard (225 kDa) on 10% gels. Substantial differences in gelatinase standard migration were noted when electrophoresis was performed under Laemmli vs modified pH conditions. Calibration graphs (Mr vs migration distance) revealed excellent correlation for gelatinase standards irrespective of electrophoretic condition (Figs. 4A and 4B). Gelatinases in Arthritic Joint Fluid
FIG. 4. Gelatinase calibration graphs under (A) Laemmli and (B) modified pH conditions. r, correlation coefficient (r Å 1.000, perfect correlation) shown respectively for graphs top to bottom. Data from Fig. 3.
in plasma (72 kDa) and PMN (92, 130, and 225 kDa) (7, 8) (Fig. 1). Gelatinase activity was confirmed as MMP since it was dependent on calcium and inhibited by 1,10-phenanthroline, a zinc chelator (Fig. 1). Effect of Gelatin Concentration Capillary gelatinases were evaluated on zymograms containing gelatin concentrations routinely used in the literature (1, 3–6). Good gelatinase activity was observed in as little as 0.25–0.5 ml capillary blood on zymograms containing 1–1.5 mg/ml gelatin (Fig. 2A). Larger samples (1 ml) were, however, required for detecting all four gelatinase standards on zymograms containing 2 mg/ml gelatin. Similar sensitivity was observed in zymograms electrophoresed under modified pH conditions (Fig. 2B). As can be seen, gelatinase standard migration was not effected by gelatin concentration. In general, we found that zymograms containing gelatin at 1.5 mg/ml gave the best gelatinase activity with the most uniform background staining. Effect of Polyacrylamide Gel Concentration The effect of polyacrylamide concentration (percentage gel) on migration of gelatinase standards was eval-
/ 6m15$$9503
04-03-96 12:27:44
abas
The utility of the standards was demonstrated relative to gelatinases in synovial fluids collected from osteo- (OA), rheumatoid (RA), and septic arthritis (SA) joints (Fig. 5). As can be seen, all three synovial fluids contained the 72-kDa gelatinase. A minor gelatinase was noted in OA between the 92- and 130-kDa standards. This unknown gelatinase at 104 kDa (calibration graph above) appeared to be present at slightly higher levels in RA. RA and SA contained substantial PMN gelatinases (92, 130, and 225 kDa) whose levels correlated with white blood cell counts of 21,000 and 450,000/ml, respectively. In addition, SA contained what appeared to be activated forms of the 92- and 72kDa gelatinases at 85 and 66 kDa, respectively (calibration graph above). CONCLUSION
We describe the use of gelatinase standards from human capillary blood for calibrating gelatin zymo-
FIG. 5. Gelatinases in arthritic joint fluid. Samples (10 ml) were analyzed on 7.5% gels containing 1.5 mg/ml gelatin. Lanes: C, capillary gelatinase standards; OA, osteo-; RA, rheumatoid; and SA, septic arthritis synovial fluid. Molecular weights (kDa) are indicated. Closed and open arrows indicate activated 92- and 72-kDa forms, respectively.
AP-Anal Bio
356
MAKOWSKI AND RAMSBY
grams. The standards are easily obtained and well characterized and should thus facilitate interlaboratory comparison and standardization.
4. Manicourt, D-H., and Lefebvre, V. (1993) Anal. Biochem. 215, 171–179.
REFERENCES
6. Kleiner, D. E., and Stetler-Stevenson, W. G. (1994) Anal. Biochem. 218, 325–329.
1. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196– 202. 2. Stetler-Stevenson, W. G. (1994) Invasion Metastasis 14, 259– 268. 3. Koolwjik, P., Miltenburg, A. M. M., van Erck, M. G. M., Oudshoorn, M., Niedbala, M. J., Breedveld, F. C., and van Hinsbergh, V. W. M. (1995) J. Rheumatol. 22, 385–393.
/ 6m15$$9503
04-03-96 12:27:44
abas
5. Schonermark, M. P., Heitefuss, P., Isernhagen, B., and Lenarz, T. (1995) Anal. Biochem. 229, 144–145.
7. Hibbs, M. S., Hasty, K. A., Seyer, J. M., Kang, A. H., and Mainardi, C. L. (1985) J. Biol. Chem. 260, 2493–2500. 8. Vartio, T., and Baumann, M. (1989) FEBS Lett. 255, 285–289. 9. Laemmli, U. K. (1970) Nature 227, 680–685. 10. Makowski, G. S., and Ramsby, M. L. (1993) Anal. Biochem. 212, 283–285.
AP-Anal Bio
236, 353–356 (1996)
0179
Calibrating Gelatin Zymograms with Human Gelatinase Standards Gregory S. Makowski* and Melinda L. Ramsby† *Department of Laboratory Medicine, †School of Medicine, MC-2235, University of Connecticut School of Medicine, Farmington, Connecticut 06030
Received December 4, 1995
We describe the use of gelatinase standards from human capillary whole blood for calibrating gelatin zymograms. Capillary blood was obtained by fingerstick puncture and prepared in nonreducing Laemmli SDS – PAGE sample buffer without heating. Gelatin zymography revealed that whole blood (0.25 – 1 ml) contained substantial fibroblast-derived (72kDa) and neutrophil-derived (92-, 130-, and 225-kDa) gelatinases which could be used for calibration purposes. Calibration was linear under different electrophoretic conditions using 6.5– 10% polyacrylamide gels containing 1 –2 mg/ml gelatin (correlation range, r Å 0.990 – 0.998; perfect correlation, r Å 1.000). The use of the standards for characterizing gelatinases from arthritic joint fluid is demonstrated. The standards are easily obtained and well characterized, and should facilitate interlaboratory comparison and standardization. q 1996 Academic Press, Inc.
Matrix metalloproteinases (MMP)1 are a diverse group of collagen-degrading enzymes implicated as playing a pivotal, although poorly defined, role in pathologic states characterized by aberrant inflammatory or invasive processes. Instrumental for elucidating the role of MMP in disease is the technique of gelatin zymography, an SDS–PAGE-based method performed with copolymerized gelatin (1). Inherent to gelatin zymography is the capacity to simultaneously detect both activated and latent forms of MMP while concomitantly resolving MMP of similar as well as diverse molecular weight (Mr). Gelatin zymography thus enables investigation of the full spectrum of MMP subclasses and iso1 Abbreviations used: MMP, matrix metalloproteinases; PMN, polymorphonuclear leukocytes; ACD, acid–citrate–dextrose; OA, osteoarthritis; RA, rheumatoid arthritis; SA, septic arthritis.
forms, the relative proportions of which appear to correlate with disease state and severity (2, 3). Despite its numerous applications, the utility of gelatin zymography is limited by poor interlaboratory comparison due to lack of a readily available MMP Mr reference standard. Thus, investigators have used no standards, standards of unclear origin, or ‘‘in-house’’ MMP purified by labor-intensive chromatographic methods (3–6). Toward the goal of defining a readily available MMP Mr standard, we investigated the feasibility of using a fingerstick blood sample as a reference material. Such samples are easily obtained and contain MMP of white blood cell and fibroblastic origin, the Mr of which have been well characterized (7, 8). MATERIALS AND METHODS
Materials Acrylamide and N,N*-methylene-bis-acrylamide were from Boehringer Mannheim Corp. (Indianapolis, IN). Vacutainer collection tubes and Flow Lancet were from Becton Dickinson Corp. (Rutherford, NJ). Ficoll–Hypaque was from LKB-Pharmacia (Piscataway, NJ). Unless specified, all other reagents were from Sigma Chemical Co. (St. Louis, MO). Methods Preparation of samples. A drop of human capillary blood (15–30 ml) was obtained by fingerstick puncture and placed in a tared polypropylene tube. The sample was weighed and 20 vol nonreducing Laemmli sample buffer (1, 9) was immediately added. The sample was then vortex mixed (30 s) and aliquots stored (0307C, stability 3–6 months). Polymorphonuclear leukocytes (PMN) were obtained from acid–citrate–dextrose (ACD) anticoagulated whole blood following dextran sedimentation, Ficoll–Hypaque centrifugation, and 353
0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ 6m15$$9503
04-03-96 12:27:44
abas
AP-Anal Bio
354
MAKOWSKI AND RAMSBY
FIG. 1. Gelatin zymography of capillary whole blood gelatinases. Zymograms were incubated in the presence (/Ca) and absence (0Ca) of calcium, and calcium plus a zinc chelator (/Ca/0Zn). Neutrophil (N) and plasma (P) gelatinases were used for comparison. Samples (15 ml) were analyzed on 7.5% gels containing 1 mg/ml gelatin (1). Molecular weights (kDa) are indicated.
hypotonic lysis (7). PMN purity routinely averaged 95– 98%. Gelatinases were released from PMN (3000/ml) by freeze–thaw in 1 M NaCl (7) and prepared as described above. Plasma was immediately obtained following centrifugation of sodium citrate anticoagulated whole blood. Synovial fluid was collected in the presence of FIG. 3. Effect of polyacrylamide concentration (6.5–10% gel) on gelatinase standard resolution under (A) Laemmli and (B) modified pH conditions. Standards (20 ml) were analyzed on zymograms containing 1.5 mg/ml gelatin. Molecular weights (kDa) are indicated.
EDTA, cell counts performed, and cleared by centrifugation (10,000g, 20 min). Plasma and synovial fluid were prepared in nonreducing sample buffer as described above. Gelatin zymography. Gelatin zymography (1) was performed on 6.5–10% polyacrylamide gels (11 cm height, 0.75 mm thickness) containing 1–2 mg/ml gelatin under Laemmli (9) or modified pH (resolving gel pH adjusted to 9.2) conditions (10). Following electrophoresis (20 mA, 257C), gels were washed twice in 200 ml 2.5% Triton X-100 (30 min each) and incubated in 100 ml 50 mM Tris–HCl, 5 mM CaCl2 , pH 7.6 (18–20 h, 377C). For inhibition studies, zymograms were incubated in the absence of calcium or in the presence of calcium plus 5 mM 1,10-phenanthroline. Gels were stained with 0.2% Coomassie brilliant blue R-250 (50% methanol, 10% acetic acid) and destained appropriately (40% methanol, 10% acetic acid). Gelatinase activity was evident as cleared (unstained) regions. RESULTS AND DISCUSSION FIG. 2. Effect of gelatin concentration (1, 1.5, and 2 mg/ml) on gelatinase standard resolution under (A) Laemmli and (B) modified pH conditions. Lanes: 1, 5; 2, 10; 3, 15; and 4, 20 ml loaded/lane (7.5% gels used). Molecular weights (kDa) are indicated.
/ 6m15$$9503
04-03-96 12:27:44
abas
Capillary Gelatinase Characterization Zymography of capillary blood demonstrated four gelatinases that migrated identically to those present
AP-Anal Bio
CALIBRATION OF GELATIN ZYMOGRAMS
355
uated (Figs. 3A and 3B). Although good activity was apparent over the percentage gel range, some obscuring was observed for the largest gelatinase standard (225 kDa) on 10% gels. Substantial differences in gelatinase standard migration were noted when electrophoresis was performed under Laemmli vs modified pH conditions. Calibration graphs (Mr vs migration distance) revealed excellent correlation for gelatinase standards irrespective of electrophoretic condition (Figs. 4A and 4B). Gelatinases in Arthritic Joint Fluid
FIG. 4. Gelatinase calibration graphs under (A) Laemmli and (B) modified pH conditions. r, correlation coefficient (r Å 1.000, perfect correlation) shown respectively for graphs top to bottom. Data from Fig. 3.
in plasma (72 kDa) and PMN (92, 130, and 225 kDa) (7, 8) (Fig. 1). Gelatinase activity was confirmed as MMP since it was dependent on calcium and inhibited by 1,10-phenanthroline, a zinc chelator (Fig. 1). Effect of Gelatin Concentration Capillary gelatinases were evaluated on zymograms containing gelatin concentrations routinely used in the literature (1, 3–6). Good gelatinase activity was observed in as little as 0.25–0.5 ml capillary blood on zymograms containing 1–1.5 mg/ml gelatin (Fig. 2A). Larger samples (1 ml) were, however, required for detecting all four gelatinase standards on zymograms containing 2 mg/ml gelatin. Similar sensitivity was observed in zymograms electrophoresed under modified pH conditions (Fig. 2B). As can be seen, gelatinase standard migration was not effected by gelatin concentration. In general, we found that zymograms containing gelatin at 1.5 mg/ml gave the best gelatinase activity with the most uniform background staining. Effect of Polyacrylamide Gel Concentration The effect of polyacrylamide concentration (percentage gel) on migration of gelatinase standards was eval-
/ 6m15$$9503
04-03-96 12:27:44
abas
The utility of the standards was demonstrated relative to gelatinases in synovial fluids collected from osteo- (OA), rheumatoid (RA), and septic arthritis (SA) joints (Fig. 5). As can be seen, all three synovial fluids contained the 72-kDa gelatinase. A minor gelatinase was noted in OA between the 92- and 130-kDa standards. This unknown gelatinase at 104 kDa (calibration graph above) appeared to be present at slightly higher levels in RA. RA and SA contained substantial PMN gelatinases (92, 130, and 225 kDa) whose levels correlated with white blood cell counts of 21,000 and 450,000/ml, respectively. In addition, SA contained what appeared to be activated forms of the 92- and 72kDa gelatinases at 85 and 66 kDa, respectively (calibration graph above). CONCLUSION
We describe the use of gelatinase standards from human capillary blood for calibrating gelatin zymo-
FIG. 5. Gelatinases in arthritic joint fluid. Samples (10 ml) were analyzed on 7.5% gels containing 1.5 mg/ml gelatin. Lanes: C, capillary gelatinase standards; OA, osteo-; RA, rheumatoid; and SA, septic arthritis synovial fluid. Molecular weights (kDa) are indicated. Closed and open arrows indicate activated 92- and 72-kDa forms, respectively.
AP-Anal Bio
356
MAKOWSKI AND RAMSBY
grams. The standards are easily obtained and well characterized and should thus facilitate interlaboratory comparison and standardization.
4. Manicourt, D-H., and Lefebvre, V. (1993) Anal. Biochem. 215, 171–179.
REFERENCES
6. Kleiner, D. E., and Stetler-Stevenson, W. G. (1994) Anal. Biochem. 218, 325–329.
1. Heussen, C., and Dowdle, E. B. (1980) Anal. Biochem. 102, 196– 202. 2. Stetler-Stevenson, W. G. (1994) Invasion Metastasis 14, 259– 268. 3. Koolwjik, P., Miltenburg, A. M. M., van Erck, M. G. M., Oudshoorn, M., Niedbala, M. J., Breedveld, F. C., and van Hinsbergh, V. W. M. (1995) J. Rheumatol. 22, 385–393.
/ 6m15$$9503
04-03-96 12:27:44
abas
5. Schonermark, M. P., Heitefuss, P., Isernhagen, B., and Lenarz, T. (1995) Anal. Biochem. 229, 144–145.
7. Hibbs, M. S., Hasty, K. A., Seyer, J. M., Kang, A. H., and Mainardi, C. L. (1985) J. Biol. Chem. 260, 2493–2500. 8. Vartio, T., and Baumann, M. (1989) FEBS Lett. 255, 285–289. 9. Laemmli, U. K. (1970) Nature 227, 680–685. 10. Makowski, G. S., and Ramsby, M. L. (1993) Anal. Biochem. 212, 283–285.
AP-Anal Bio