Figure 1.
Influence of pH on zymogen proFheCL1 autocatalytic activation and activity of mature FheCL1.
(A) Analysis by SDS-PAGE of the activation of 0.2 mg/ml FheproCL1 to mature FheCL1 in 0.1 M sodium acetate buffer, pH 4.5. The zymogen, mature enzyme and degraded prosegment are indicated by arrowheads. (B) Kinetic study of the activation of 5 nM FheproCL1 between pH 4.0 and pH 7.0 in the presence of 2 µM Z-Phe-Arg-NHMec. (C) Relative kcat/Km values for the hydrolysis of 0.5 µM Z-Phe-ArgN-Mec by 0.14 nM mature FheCL1 at 37°C.
Figure 2.
Stability of the zymogen proFheCL1Gly25 and mature FheCL1 at various pH values.
(A) Far-UV CD spectra of 5.3 µM FheproCL1Gly25 in 50 mM sodium acetate buffer, pH 4.0 and in 50 mM sodium phosphate buffer, pH 7.5. (B) Enzymatic stability of 6.0 µM mature FheCL1 at 37°C and in 0.1 M buffers over the pH range 2.5–9.0. Enzyme activity was monitored at various time-points by diluting aliquots of the reactions into 0.1 M sodium acetate buffer, pH 5.5, containing 3 mM DTT before addition of 5 µM Z-Phe-Arg-NMec.
Figure 3.
Effect of small molecular thiols on the activity of FheCL1.
FheCL1 was incubated with (A) DTT, (B) GSH and (C) l-cysteine before adding the fluorogenic substrate Z-Phe-Arg-NHMec. Final assays contained 4 nM enzyme, (10 nM–10 mM) reducing agent and 5 µM substrate in 0.1 M sodium acetate buffer, pH 4.5, with 1 mM EDTA.
Figure 4.
pH dependency of FheCL1 hydrolytic activity against protein substrates Hb and ovalbumin.
(A) Hb incubated alone in solutions buffered in the ranges pH 3.5–pH 8.0; (B) Hb incubated with FheCL1 in the same buffers at pH 3.5–pH 8.0; (C) Ovalbumin incubated alone in solutions buffered in the ranges pH 3.5–pH 8.0, and (D) Ovalbumin incubated with FheCL1 in the same buffers at pH 3.5–pH 8.0. Digests were analysed by 15% SDS-PAGE. Molecular size markers are indicated on the left.
Figure 5.
Regulation of FheCL1 hydrolytic activity against haemoglobin by pH.
(A) Spectra of 5.0 µM Hb following 1 hr incubation in 0.1 M buffer at pH 3.5, pH 4.0, pH 5.5 and pH 7.0. Decreases in the Soret peak absorbance at 414 nm shows Hb denaturation with decreasing pH. (B) Progress of denaturation of 5.0 µM haemoglobin at several pH values over time as revealed by the decrease in absorbance at 414 nm. (C) Susceptibility of Hb to FheCL1 hydrolytic activity at pH 4.0 and pH 7.0 in the presence 1 mM GSH. (a) 5.0 µM Hb and 1 mM GSH at pH 7.0 (b) 5.0 µM Hb, 1 mM GSH and 1 µM FheCL1 at pH 7.0 (c) 5.0 µM Hb and 1 mM GSH at pH 4.5 (d) 5.0 µM Hb, 1 mM GSH and 1 µM FheCL1 at pH 4.5.
Figure 6.
Characterisation of hydrolytic activity of FheCL1 on Hb.
(A) Progress of digestion of Hb by recombinant FheCL1. Purified haemoglobin (Hb, lane 1) was digested by FheCL1 in 0.1 M sodium acetate buffer, pH 4.0, containing 1 mM GSH and 1 mM EDTA at 37°C. Reactions were stopped at time 0 and at various time-points (indicated on x axis) by the addition of the cysteine protease inhibitor E-64 and analysed on 4–12% Bis-Tris NuPage gels. The arrow indicates the position of FheCL1 (25 kDa) that was not degraded in the reaction. Molecular mass markers are shown on the left. (B) Map of Hb α- and β-chains indicating sites of FheCL1 cleavage the substrates. Cleavage sites within Hb present in 10 min reactions (arrows) compared to cleavages that occur with longer incubation times (120 min, arrowheads) as determined by nanoLc-MS/MS.
Figure 7.
Analysis of peptides released from Hb following digestion by FheCL1.
Frequency (expressed as a percentage) of peptides of varying length released following proteolysis of Hb alpha and beta chains by FheCL1.
Figure 8.
P2 residues in peptides released from Hb following digestion by FheCL1.
Analysis of frequency by which amino acids occur at the P2 position from the peptide bonds cleaved by FheCL1 in Hb α- and β-chains (corresponding to the 10 min reactions shown in Figure 6). The y axis represents the frequency of a particular residue at the P2 position of the haemoglobin substrates and the x axis shows the amino acids as represented by the one-letter code.