High Pressure Liquid Chromatography HPLC

1. Different GAG-charged matrices were also packed into a 3-mL stainless steel column and equilibrated with 20 mM Tris-HCl, pH 7.5, at 0.5 mL/min using a Waters HPLC system (see Figs. 2 and 3).

Fig. 1. Elution profile of the apoSAAl.1 CNBr peptides on a heparin-Affigel column: testing apoSAAl.l CNBr fragments for heparin-binding activity. ApoSAAl.1 CNBr cleavage fragments are shown above the graph; 16mer (residues 1-16), 7mer (residues 17-23), 53mer (residues 24-76), and 27mer (residues 77-103). A heparin-binding consensus sequence, (XBBXBX, X = nonbasic, B = basic) is underlined in m27mer.

Fig. 1. Elution profile of the apoSAAl.1 CNBr peptides on a heparin-Affigel column: testing apoSAAl.l CNBr fragments for heparin-binding activity. ApoSAAl.1 CNBr cleavage fragments are shown above the graph; 16mer (residues 1-16), 7mer (residues 17-23), 53mer (residues 24-76), and 27mer (residues 77-103). A heparin-binding consensus sequence, (XBBXBX, X = nonbasic, B = basic) is underlined in m27mer.

2. Samples (20-80 ^g in 150-200 ^L) were injected onto the column, washed with 3 bed volumes (18 min) of the same buffer, and then developed with a 0-1.0 M NaCl linear gradient for 10 bed volumes (60 min). The eluate was monitored continuously at 214 nm and the absorbance plotted against the retention time (RT).

3. Generally, unbound peptides/proteins eluted 6.5-7.0 min after loading and, based on the RTs for the bound peptides/proteins, the NaCl concentration at which desorption took place was calculated; desorption [NaCl] = (RT -6.5 min -18 min) / 60 min.

3.5. ApoSAA2 Peptides

3.5.1. CNBr Peptides

1. ApoSAA1.1 was cleaved at Met-X peptide bonds with cyanogen bromide (CNBr). Protein was dissolved in 70% formic acid at 1 mg/mL, to which 5.5 mg/mL CNBr was added (250 molar excess over Met) plus freeL-Trp (5 molar excess over Met) to protect Trp residues.

2. The reaction was carried out under nitrogen, at room temperature overnight, and then the solvent was evaporated by vacuum centrifugation.

3. The reaction was evaluated and peptides purified, by reverse-phase (RP)-HPLC. A semipreparative C-18 Vydac column was equilibrated with 10% acetonitrile, 0.1%

8384 86 89 95 102

ADQEANRHGRSGKDPNYYRPPGLPAKY

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 Retention time (min.)

Fig. 2. Identification of the basic residues of m27mer that are important for heparin binding. Chromatography was performed on heparin-Sepharose 4B and the elution profiles were similar to that seen with heparin-Affigel (not shown) with the exception of K102-. Carbodiimide treatment of HS interferred with binding (HS-Sepharose + carbodiimide). The sequence of m27mer is shown above the graph.

TFA. Peptides were dissolved in 40% formic acid, filtered through a 0.2-^m filter, or centrifuged at 10,000g, and loaded onto the column, washed for 5 min at 3 mL/min, then developed with a 1.5%/min acetonitrile linear concentration gradient for 30 min, followed by 4.3%/min acetonitrile for 10 min, bringing the elution buffer to 98% acetonitrile by 45 min.

4. The separated peptides were identified by amino-terminal sequencing and molecular-weight determination by mass spectroscopy carried out at the Alberta Peptide Institute (Edmonton, Alberta, Canada).

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