ANALYTICAL BIOCHEMISTRY

Analytical Biochemistry 326 (2004) 276277 www.elsevier.com/locate/yabio

Notes & Tips

High-performance liquid chromatography-based protease detection at the picogram level

Edward J. Bures, Christine C. Siska, and Andrei A. Raibekas*
Department of Pharmaceutics, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA Received 22 August 2003

Highly sensitive detection of proteolytic activity can be achieved by means of uorophore-tagged articial substrates. Protease-catalyzed cleavage of the substrate leads to a quantitative release of an uncoupled uorophore and a corresponding increase in uorescence [1 7]. Typically, the protease/substrate mixture in an assay buer solution is incubated at 2537 C with uorescence intensity of the reaction product measured in a suitable uorometer. The procedure, however, can become quite tedious when protease detection requires a large number of samples and prolonged incubation time. In addition, the uorescent intensity parameter alone may not be signicant enough to provide statistically valid and reproducible data in the lower, close to the background, detection range. In this report we describe a new procedure for protease detection utilizing a semiautomated, HPLC-based technique. Trypsin served as a model proteolytic enzyme. The rhodamine-conjugated substrate was chosen for its stability, low uorescence background, and high sensitivity [5,6]. The developed procedure required only about 5 min per analysis and minimal labor and allowed detection of a protease concentration at the picogram level. Reagents and solvents were of the highest available purity. The rhodamine 110, bis-(Cbz)-L -isoleucyl-L prolyl-L -arginine amide, dihydrochloride [(Cbz-Ile-ProArg-NH)2 -rhodamine] was purchased from Molecular Probes (Eugene, OR). Sequencing-grade trypsin was obtained from Roche (Indianapolis, IN). The HPLC system used was an Agilent 1100 (Palo Alto, CA) equipped with on-line uorescence detection and a temperature-controlled 100-well sample tray. Reversedphase chromatography was performed on a Proteo (90 A, 4-lm) reversed-phase HPLC column (4.6 50 mm;
* Corresponding author. Fax: 1-805-375-5794. E-mail address: andreir@amgen.com (A.A. Raibekas).

Phenomenex, Torrance, CA). The ow rate was 1 ml/ min. Solvent A consisted of 0.1% triuoroacetic acid (v/ v) in water and solvent B was 90% acetonitrile, 0.1% triuoroacetic acid (v/v) in water. The column was preequilibrated with 40% B. The sample was loaded onto the column and the elution was performed immediately at 100% B for 3 min followed by a 2-min reequilibration step at 40% B. The sample injection volume was 100 ll and the volume of the uorescence detector ow cell was 4 ll. The column was maintained at a temperature of 50 C and the sample tray was set at 30 C. The uorescence detector settings were adjusted to provide an excitation at 498 nm and to monitor an emission at 521 nm. Reactions were performed in 25 mM phosphate buer, pH 7.6, containing 25 mM L -arginine, 1% sucrose, and 100 mM NaCl. Usually, 0.9 ml volume of 1.14 lM (Cbz-Ile-Pro-Arg-NH)2 -rhodamine solution in reaction buer was mixed with 0.1 ml volume of a protease-containing solution in the same buer. The sample was placed in a 1.5-ml reaction vial (National Scientic Co., Duluth, GA) that was immediately capped and transferred into the sample tray. The incubation was conducted at 30 C in the sample tray while 100-ll aliquots were automatically withdrawn and analyzed at preset timed intervals. Processing of the resulting chromatograms was automated to integrate the peak area in the retention time range from 0.8 to 1.2 min. Chromatography conditions were established to monitor an accumulation of a single uorescent peak (i.e., released rhodamine) resulting from the cleavage of the (Cbz-Ile-Pro-Arg-NH)2 -rhodamine with trypsin (Fig. 1). The integrated peak area values were calculated and plotted against the corresponding trypsin concentrations. As illustrated in Fig. 1 (lane 5), in our procedure, we were able to detect trypsin levels as low as 40 pg/ml after 3 h of incubation at 30 C and pH 7.6. The range of detection allowed measurements within two

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Notes & Tips / Analytical Biochemistry 326 (2004) 276277

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Fig. 1. HPLC uorescence data analysis of samples containing various concentrations of trypsin after 3-h incubation with (Cbz-Ile-Pro-Arg-NH)2 rhodamine at 30 C and pH 7.6. The trypsin concentrations in incubation mixture are as follows: 0, 5, 10, 20, 40, 80, 156, and 312 pg/ml (lanes 18). The inset shows a double logarithmic plot of the integrated peak areas as a function of trypsin concentration in incubation mixture. The detection range derived from the plot is of 50 to 5000 pg/ml.

orders of magnitude of trypsin concentration (from 50 to 5000 pg/ml) as judged from a linear region of the saturation curve (Fig. 1, inset). In the next step, the assay was applied to analyze three dierent lots of the mammalian cell-derived, puried recombinant protein rAIF2 for protease contamination. Samples were prepared using 50 mg/ml protein stock solution and analyzed as described above during 80 h incubation in the sample tray at 30 C. The assay detected a trace serine protease activity in one of the purication lots (Fig. 2, lot 1). This was in agreement with SDSPAGE analysis where only lot 1 revealed protein degradation. It also must be noted (Fig. 2) that lots 2 and 3 showed an increase in the background signal similarly to blank that can be attributed to a nonenzymatic conversion of a small fraction of the substrate into a product.

To conclude, we developed a highly sensitive reversed-phase HPLC-based procedure for quantitative proteolytic enzyme detection as demonstrated with trypsin. Although we used 100 ll sample volume per single injection and analysis, it can be scaled down further depending on the sample requirements. The procedure is not limited to trypsin alone and can be potentially adapted to any type of protease by choosing an appropriate rhodamine-linked substrate and reaction conditions. Finally, the use of the temperature-controlled sample tray as an integral part of the HPLC system minimizes time and handling during sample incubation and analysis.

References
[1] M. Zimmerman, B. Ashe, E.C. Yurewicz, G. Patel, Sensitive assays for trypsin, elastase, and chymotrypsin using new uorogenic substrates, Anal. Biochem. 78 (1977) 4751. [2] W. Nieuwenhuizen, G. Wijngaards, E. Groeneveld, Flourogenic peptide amide substrates for the estimation of plasminogen activators and plasmin, Anal. Biochem. 83 (1977) 143148. [3] P.A. Pierchala, C.P. Dorn, M. Zimmerman, A new uorogenic substrate for plasmin, Biochem. J. 183 (1979) 555559. [4] M. Suzuki, T. Ueno, T. Takahashi, Y. Kanaoka, T. Okuyama, H. Furuya, T. Sekine, A new uorometric ultramicro determination of serum cysteine aminopeptidase using an aminocoumatine derivative, Clin. Chim. Acta 115 (1981) 223228. [5] S.P. Leytus, L.L. Melhado, W.F. Mangel, Rhodamine-based compounds as uorogenic substrates for serine proteinases, Biochem. J. 209 (1983) 299307. [6] S.P. Leytus, W.L. Patterson, W.F. Mangel, New class of sensitive and selective uorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine, Biochem. J. 215 (1983) 253260. [7] H. Hug, M. Los, W. Hirt, K.-M. Debatin, Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells, Biochemistry 38 (1999) 1390613911.

Fig. 2. Comparative analysis of the rAIF2 protein purication lots. The relative change in the integrated uorescent peak area is plotted against incubation time at 30 C. The data for lots 1, 2, and 3 are shown as dark circles, open circles, and dark triangles, respectively. The buer alone (blank) data are shown as open triangles.