Stability of HAMLET--a kinetically trapped alpha-lactalbumin oleic acid complex

Abstract

The stability toward thermal and urea denaturation was measured for HAMLET (human alpha-lactalbumin made lethal to tumor cells) and alpha-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than alpha-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15 degrees C lower for apo HAMLET than for apo alpha-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.

Figure 1.

(A) Human α-lactalbumin (PDB accession no. 1B90). The sphere represents a calcium ion; disulphide bridges are drawn as black lines. The C and N termini and helix A–C are indicated. (B) Space filling and line representation of oleic acid (OA) (same scale as α-lactalbumin). A and B were prepared using MOLMOL (Koradi et al. 1996) and rendered in POV-Ray. (C) Amino acid sequences of human and bovine α-lactalbumin. The bovine sequence is only shown at positions that differ from the human protein. The secondary structure elements as shown in A are marked. Calcium site residues (79–88) and disulphide bridges are indicated.

Figure 2.

Thermal denaturation monitored by far- and near-UV CD. Data are shown as mean residue molar ellipticity. Temperature scan between 5° and 90°C monitored at 270 nm (A–C) and 222 nm (D–F) for human α-lactalbumin (dashed line), HAMLET (solid line), in 5 mM Tris/HCl, 0.15 M NaCl (pH 7.4) with 0.5 mM EDTA (A,D), 1 mM CaCl₂ (B,E) or 10 mM CaCl₂ (C,F). Data for the molten globule form at pH 2.0, 0.15 M NaCl, are included in A and D (dotted line).

Figure 3.

Thermal denaturation monitored by far- and near-UV CD for HAMLET (solid line) and human α-lactalbumin (dashed line). (A) Examples of curves fitted to data in 0.5 mM EDTA. (B) Normalized data in

Figure 4.

Near-UV CD spectra (320–250 nm) at 10°C intervals from 5° to 55°C in 5 mM Tris/HCl, 0.15 M NaCl (pH 7.4), with EDTA or calcium as indicated below. Data are shown as mean residue molar ellipticity. Human α-lactalbumin in 1 mM CaCl₂ (A), 1 mM EDTA (C), and HAMLET in 0.6 mM CaCl₂ (B) and 1 mM EDTA (D). A specrum of HAMLET in 0.6 mM CaCl₂ after denaturation and cooling to 5°C is shown in B as a thin line with symbols.

Figure 5.

Differential scanning calorimetry (DSC) from 5° to 95°C for 70 μM HAMLET (solid line) or human α-lactalbumin (dashed line) in 1 mM EDTA (A), 0.5 (HAMLET) or 1 (α-lactalbumin) mM Ca²⁺ (B), 10 mM Ca²⁺ (C), 0.15 M NaCl and 1 mM EDTA (D), 0.15 M NaCl and 0.5 (HAMLET) or 1 (α-lactalbumin) mM Ca²⁺ (E). Thermograms of 7–350 αM HAMLET in 0.15 M NaCl and 1 mM Ca²⁺ are shown in F. All samples contain 5 mM Tris/HCl (pH 7.5).

Figure 6.

Urea denaturation of HAMLET (triangles) and α-lactalbumin (circles) monitored by fluorescence at 335 nm (open symbols) and CD signal at 222 nm (filled symbols), in 5 mM Tris/HCl, 0.15 M NaCl (pH 7.4), with 0.5 mM EDTA (A) or 1 mM CaCl₂ (B). The CD data is normalized to apparent fraction unfolded (F_app). The fluorescence data in CaCl₂ is normalized to Fapp, but in EDTA the fluorescence intensity is shown. Each presented data set is an average over two to four experiments.

Figure 7.

Apoptosis bioassay. (A) Cell viability and (B) DNA fragmentation of the L1210 cancer line after exposure to heat-treated (room temperature, 37°, 50°, or 75°C) aliquots of HAMLET. The apoptotic effect is decreased with the temperature, and at 75°C, no activity was left. The control α-lactalbumin did not affect the cells at all.

Figure 8.

Titration of apo α-lactalbumin with oleic acid as followed by ¹H-NMR. One (1) mM protein was dissolved in D₂O (pH 7.0). Oleic acid was titrated in steps of 0.25 equivalents at 37°C. One signal appears at 6.8 ppm and reaches saturation, while another signal appears later in the titration at 5.3 ppm and grows linearly after an initial delay (signals indicated by arrows).

Figure 9.

Schematic reaction pathway for HAMLET denaturation. When HAMLET is denatured, oleic acid is released and denatured α-lactalbumin is obtained (arrow 1). The denaturation equilibrium is then established for 3-lactalbumin (arrow 2).