1997, 272, 31293-31300. (9) Grant, D. A. W.; Hermon-Taylor, J. Biochim. Biophys. Acta 1979, 567, 207-215. Enteropeptidase: Trypsinogen ---â Trypsin. Trypsin:.
Protection against Proteolytic Cleavage by Compatible Solutes Sonja Kolp,1 Markus Pietsch,1 Erwin A. Galinski2 and Michael Gütschow1 1 Pharmaceutical
of Microbiology and Biotechnology, University of Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany Enteropeptidase:
Introduction
Trypsinogen
Serine proteinases are involved in several physiological and pathophysiological processes. It is characteristic of these proteolytic enzymes that they are synthesized as inactive precursors, termed zymogens, being activated autocatalytically or by other proteinases.
Trypsin:
Some compatible solutes (1) stabilize proteins and protect them against proteolytic cleavage (2-5). It was demonstrated that proteins preferentially exclude compatible solutes from their vicinity due to a higher affinity for water. Preferential hydration of proteins in the presence of compatible solutes leads to an increase in stability and maintenance of enzymatic activity (6). The unfavorable transfer of the peptide backbone from water to compatible solute solution is the dominant factor for the stabilizing effect (7). As shown by the aforementioned studies, the presence of some compatible solutes opposes the enlargement of surface area, prevents unfolding and favors a more compact protease-resistant conformation. The fact that compatible solutes have individual characteristics regarding their interactions with the surfaces of different proteins is in agreement with our results.
Scheme 1: Zymogen activation
We have studied the effects of the compatible solutes betaine, ectoine and hydroxyectoine (Scheme 3) on the enzymatic activation of the zymogens trypsinogen and chymotrypsinogen (Scheme 1). Activity was measured spectrophotometrically in coupled assays (8) based on the cleavage of the chromogenic substrates catalyzed by activated trypsin and chymotrypsin, respectively. Separately, the influence of solutes on the enzyme-catalyzed cleavage of low-molecular weight substrates (9) was determined (Table 1, Scheme 2). The ability of compatible solutes to preserve enzymatic activity during storage in solution was also investigated.
betaine
Chymotrypsin: Suc-Ala-Ala-Pro-Phe-p-nitroanilide
COO
-
H3C
ectoine
azo compound
Scheme 2: Spectrophotometric assays
300
Figure 1: Betaine
N H
COO-
hydroxyectoine
100
0
Table 1: Influence of solutes (800 mM) on enzyme-catalyzed cleavage of lowmolecular weight substrates shown in Scheme 2 betaine 93% 108% 94%
ectoine 76% 101% 34%
3
0
1
40 time (min)
60
Assay: 25 °C, 405 nm 200 IM Suc-Ala-Ala-Pro-Phe-pNA 20 mM Tris-HCl, 150 mM NaCl, pH 8.4 6 % DMSO 950 Il + 50 Il from incubation Incubation: 25°C 6.25 Ig/ml chymotrypsinogen 0.625 Ig/ml trypsin 800, 400, 100 mM ectoine or H2O (control) 20 mM Tris-HCl, 150 mM NaCl, pH 8.4 6 % DMSO
0 0
200
400
600
300
Figure 3: Hydroxyectoine
800
200
100
40 time (min)
60
200
100
0 20
40 time (min)
60
Assay: 25 °C, 405 nm 200 IM Suc-Ala-Ala-Pro-Arg-pNA 20 mM Tris-HCl, 150 mM NaCl, pH 8.4 6 % DMSO 950 Il + 50 Il from incubation
20
40 time (min)
60
Incubation: 25°C 12.5 Ig/ml trypsinogen 0.98–1.96 Ig/ml enteropeptidase 800, 400, 100 mM solute or H2O (control) 20 mM sodium citrate, pH 5.6 6 % DMSO
Figure 1- 3: Trypsinogen activation in the presence of solutes 120
Open circles: control Closed circles: 800 mM ectoine
100
activity (%)
activity (%)
rate (%/min)
hydroxyectoine 93% 116% 72%
100
20
Figure 2: Ectoine
0
Open circles: control Closed circles: 100 mM solute Open squares: 400 mM solute Closed squares: 800 mM solute
Insert: Open circles: control Closed circles: 100 mM ectoine Open squares: 400 mM ectoine Closed squares: 800 mM ectoine
200
2
300
200
20
control 100% 100% 100%
H-Gly-(Asp)4–Lys-OH + -naphthylamine NaNO2, HCl, N-(1-naphthyl)-ethylenediamine
Scheme 3: Structures of betaine, ectoine and hydroxyectoine
enzyme trypsin chymotrypsin enteropeptidase
Suc-Ala-Ala-Pro-Phe-OH + p-nitroaniline
H-Gly-(Asp)4-Lys- -naphthylamide
activity (%)
N H
H3C
Suc-Ala-Ala-Pro-Arg-OH + p-nitroaniline
+
+
CH3
Suc-Ala-Ala-Pro-Arg-p-nitroanilide
activity (%)
H3C
COO
Chymotrypsin
Trypsin:
OH
HN
HN
-
Chymotrypsinogen
Enteropeptidase:
Enzymological investigations
H3C + N
Trypsin
activity (%)
2 Institute
Institute, Poppelsdorf, University of Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany
60
Assay: 25 °C, 405 nm 200 IM Suc-Ala-Ala-Pro-Phe-pNA 20 mM Tris-HCl, 150 mM NaCl, pH 8.4 6 % DMSO 900 Il + 100 Il from incubation
40
Incubation: 25°C
80
200 ng/ml chymotrypsin 800 mM ectoine or H2O (control)
20
20 mM Tris-HCl, 150 mM NaCl, pH 8.4 6 % DMSO
0 0
[ectoine] ( M)
80
160 time (min)
240
Figure 4: Chymotrypsinogen activation in the presence of ectoine (Data for betaine and hydroxyectoine are not shown.)
Summary It has been proposed that an increase in stability of a protein occurs in the presence of compatible solutes. We report on protection against proteolysis by the solutes ectoine and betaine supporting this proposition. Except for enteropeptidase activity, hydroxyectoine neither affected the enzyme assays (Table 1) nor protected trypsinogen (Fig. 3) or chymotrypsinogen (data not shown) against activation. In contrast, high concentrations of betaine reduced the trypsinogen activation (Fig. 1) as well as chymotrypsinogen activation (data not shown). However, protease-catalyzed cleavage of lowmolecular weight substrates was not impaired by betaine (Table 1). Our results demonstrated that ectoine drastically inhibited the cleavage of trypsinogen (Fig. 2) and chymotrypsinogen (Fig. 4). In separate assays, high concentration of ectoine also influenced the activity of trypsin and enteropeptidase by perturbing the cleavage of small substrates (Table 1). Above all, this compatible solute prevented the complete loss of chymotryptic (Fig. 5) and tryptic activity (data not shown) during storage in solution. This feature was exclusively observed in the presence of ectoine, while the other two solutes did not show any stabilizing effect.
Figure 5: Stability of chymotrypsin in the presence of ectoine (800 mM) Acknowledgement We are grateful to Bitop AG, Witten, Germany for providing ectoine and hydroxyectoine.
References (1) da Costa, M. S.; Santos, H.; Galinski, E. A. Adv. Biochem. Eng. Biotechnol. 1998, 61, 117-153. (2) Galinski, E. A.; Kaufmann, M.; Schwarz, T.; Vangala, M.-D. Eur. Pat. Appl. 2000, EP 1125583. (3) Yancey, P. H.; Siebenaller J. F. J. Exp. Biol. 1999, 202, 3597-3603. (4) Ratnaparkhi, G. S.; Varadarajan, R. J. Biol. Chem. 2001, 276, 28789-28798. (5) Reid, J.; Shelly, S. M.; Watt, K.; Price, N. C.; McEwan, I. J. J. Biol. Chem. 2002, 277, 20079-20086. (6) Timasheff, S.N.; A physicochemical basis for the selection of osmolytes by nature. In: Somero, G. N. (Ed.) Water and life. Springer-Verlag, New York, 1992, pp 70-84. (7) Liu, Y.; Bolen, D.W. Biochem. 1995, 34, 12884-12891. (8) Lu, D.; Yuan, X.; Zheng, X.; Sadler, J. E. J. Biol. Chem. 1997, 272, 31293-31300. (9) Grant, D. A. W.; Hermon-Taylor, J. Biochim. Biophys. Acta 1979, 567, 207-215.
