Biological functions of the disulfides in bovine pancreatic deoxyribonuclease

Abstract

We characterized the biochemical functions of the small nonessential (C101-C104) and the large essential (C173-C209) disulfides in bovine pancreatic (bp) DNase using alanine mutants [brDNase(C101A)] and [brDNase(C173A) and brDNase(C209A)], respectively. We also characterized the effects of an additional third disulfide [brDNase(F192C/A217C)]. Without the Ca(2+) protection, bpDNase and brDNase(C101A) were readily inactivated by trypsin, whereas brDNase(F192C/A217C) remained active. With Ca(2+), all forms of DNase, except for brDNase(C101A), were protected against trypsin. All forms of DNase, after being dissolved in 6 M guanidine-HCl, were fully reactivated by diluting into a Ca(2+)-containing buffer. However, when diluted into a Ca(2+)-free buffer, bpDNase and brDNase(C101A) remained inactive, but 60% of the bpDNase activity was restored with brDNase(F192C/A217C). When heated, bpDNase was inactivated at a transition temperature of 65 degrees C, brDNase(C101A) at 60 degrees C, and brDNase(F192C/A217C) at 73 degrees C, indicating that the small disulfide, albeit not essential for activity, is important for the structural integrity, and that the introduction of a third disulfide can further stabilize the enzyme. When pellets of brDNase(C173A) and brDNase(C209A) in inclusion bodies were dissolved in 6 M guanidine-HCl and then diluted into a Ca(2+)-containing buffer, 10%-18% of the bpDNase activity was restored, suggesting that the "essential" disulfide is not absolutely crucial for enzymatic catalysis. Owing to the structure-based sequence alignment revealing homology between the "nonessential" disulfide of bpDNase and the active-site motif of thioredoxin, we measured 39% of the thioredoxin-like activity for bpDNase based on the rate of insulin precipitation (DeltaA650nm/min). Thus, the disulfides in bpDNase not only play the role of stabilizing the protein molecule but also may engage in biological functions such as the disulfide/dithiol exchange reaction.

Figure 1.

SDS-PAGE analysis of the purified proteins. Each lane contained 10 μg of total protein. Lanes 1–6: bpDNase, brDNase(wt), brDNase(F192C/A217C), brDNase(C101A), brDNase(C173A), and brDNase(C209A), respectively. The gel was run under nonreducing conditions and was silver-stained.

Figure 2.

Thermal stability. (A) Purified DNases in 50 mM Tris-HCl (pH 7.5) were heated at the specified temperature for 10 min and cooled immediately on ice. The activity was measured using the hyperchromicity assay. (B) First derivative of the relative DNase activities shown in (A) to indicate the melting temperature for each DNase at the curve peak. (Filled circles) bpDNase, (hollow circles) brDNase(F192C/A217C), (filled triangles) brDNase(C101A), (hollow triangles) brDNase(C173A), (squares) brDNase(C209A).

Figure 3.

Reactivation of the guanidine-HCl-treated DNases. Purified DNases (2.5–9 μg) in 20 μL of 6 M guanidine-HCl was incubated for 30 min at 25°C and then diluted 10-fold into 100 mM Tris-HCl (pH 7.0) with 10 mM EDTA or 10 mM CaCl₂. At selected time intervals, 10-μL aliquots were removed for measurements of DNase activities using the hyperchromicity assay. (Filled circles) bpDNase with Ca²⁺, (hollow circles) bpDNase with EDTA, (filled triangles) brDNase(F192C/A217C) with Ca²⁺, (hollow triangles) brDNase(F192C/A217C) with EDTA, (filled squares) brDNase(C101A) with Ca²⁺, (hollow squares) brDNase(C101A) with EDTA, (filled down triangles) brDNase(C173A) with Ca²⁺, (hollow down triangles) brDNase(C173A) with EDTA, (hollow diamonds), brDNase (C209A) with Ca²⁺, (filled diamonds) brDNase(C209A) with EDTA.

Figure 4.

Calcium protection against trypsin inactivation. (A) The inactivation kinetics. The mixture (150 μL) containing 15–55 μg/mL DNase and 1.5–5.5 μg/mL trypsin in 50 mM Tris-HCl (pH 8.0) was incubated at 25°C. (a) with 10 mM EDTA; (b) with 10 mM CaCl₂. At selected time intervals, 10-μL aliquots were removed for measurements using the hyperchromicity assay. (Filled circles) bpDNase, (open circles) brDNase(F192C/A217C), (filled triangles) brDNase(C101A), (open triangles), brDNase(C173A), (squares), brDNase(C209A). (B) SDS-PAGE analyses of the trypsin-treated samples. The protein (1 μg) in 15 μL of 50 mM Tris-HCl (pH 8.0) was incubated for 10 min with the combination of 0.1 μg of trypsin, 10 mM CaCl₂, or 10 mM EDTA. Prior to loading, all samples were treated with β-mercaptoethanol. Gel was stained with Coomassie blue. The arrows indicate the N-terminal fragments of cleaved-bpDNase in lane 3, and brDNase(C101A) in lanes 8,9.

Figure 5.

The thioredoxin-like activity of bpDNase. (A) The dithiothreitol-dependent reduction of insulin by thioredoxin or bpDNase. The reaction mixture contained, in a final volume of 0.6 mL: 0.1M potassium phosphate (pH 7.0), 2 mM EDTA, 0.13 mM bovine insulin, 0.33 mM dithiothreitol, and 1.95 μM thioredoxin or DNases. (Open squares) Thioredoxin, (filled squares) bpDNase plus 5 mM Ca²⁺, (open circles) bpDNase, (filled circles) brDNase(F192C/A217C), (open triangles) brDNase(C101A), (filled triangles) control, without thioredoxin or DNase. (B) NADPH oxidation catalyzed by the thioredoxin/thioredoxin reductase system. The assay mixture was the same as in (A) except for NADPH (0.4 mM) replacing dithiothreitol. The reaction was started by addition of 6.95 × 10⁻⁸ M thioredoxin reductase. The NADPH oxidation was measured by recording the increase of absorbance at 340 nm until turbidity appeared. The cuvettes then were shifted as indicated by an arrow, and the increase of absorbance at 650 nm was followed to obtain the rate of precipitation. (Open circles) Thioredoxin, (filled triangles) bpDNase, (open triangles) control, without thioredoxin or DNase.

Figure 6.

Ribbon diagram for bpDNase based on the crystal structure (Oefner and Suck 1986). The structure illustrates the two intrinsic disulfides (Cys101–Cys104 and Cys173–Cys209) and the two Ca²⁺-binding sites. The newly engineered third disulfide, Cys192–Cys217, not shown in the structure, corresponds to Phe192 and Ala217, whose side chains are as shown. The distance between the guanidinium group of Arg187 and the β-carboxyl group of Asp198 is 3.05 Å. N′ and C′ indicate the NH₂- and COOH-termini, respectively.