Comparison of methods for the purification of alpha-1 acid glycoprotein from human plasma

Abstract

Alpha-1 acid glycoprotein (AGP) is a highly glycosylated, negatively charged plasma protein suggested to have anti-inflammatory and/or immunomodulatory activities. Purification of AGP could be simplified if methods that exploit its high solubility under chemically harsh conditions could be demonstrated to leave the protein in its native conformation. Procedures involving exposure of AGP to hot phenol or sulphosalicylic acid (SSA) were compared to solely chromatographic methods. Hot phenol-purified AGP was more rapidly cleared from mice in vivo following intravenous injection than chromatographically purified AGP. In contrast, SSA-purified AGP demonstrated an identical in vivo clearance profile and circular dichroism spectrum to chromatographically purified AGP. Similarly, no differences in susceptibility to enzymatic deglycosylation or reactivity with Sambucus nigra lectin were detected between AGP purified via the two methods. Incorporation of the SSA step in the purification scheme for AGP eliminated the need for a large (4 mL resin/mL of plasma) initial chromatographic step and simplified its purification without causing any detectable distortion in the conformation of the protein. Confirmation that this procedure is nondenaturing will simplify AGP purification and investigation of its possible biological roles in laboratory animals.

Figure 1

Comparison of AGP purified by solely chromatographic Method 1 or hot phenol-incorporating Method 2. (a) shows Coomassie-stained SDS reducing gels of equivalent (5 μg total protein/lane) samples of AGP preparations after initial (Cibacron Blue dye column flow-through, Affi-Blue FT, or hot phenol aqueous phase, Phenol sup.) or secondary hydroxyapatite chromatography (HT), with the Method number given beneath, and the position of molecular mass markers indicated between (in kDa) the gel images. (b) shows the percentage of the injected dose remaining in mice following injection of radioiodinated AGP purified by either Method 1 or Method 2 (n = 6, mean ± SD) versus time. (c) is identical to (b), except that the first hour post-injection is expanded to allow visualization of initial time points.

Figure 2

Reaction of AGP with neuraminidase and reaction of Method 1 versus Method 2 AGP with lectins and antibodies. AGP purified by Method 2 (AGP; 2 μg total protein/lane) was treated with or without neuraminidase (neu-AGP) and examined on SDS reducing gels or replica SNA blots (a). Molecular mass markers (in kDa) are shown at left. (b) shows 0.25–1.0 μg AGP from Methods 1 or 2 on blots probed either with Sambucus nigra agglutinin (SNA Blot) or with Anti-AGP antibodies. The SNA: anti-AGP ratio of the band intensities is shown beneath the lanes.

Figure 3

Comparison of AGP purified by solely chromatographic Method 3 or sulphosalicylic acid-incorporating Method 4. (a) and (b) show Coomassie-stained SDS reducing gels of equivalent (5 μg total protein/lane) samples of AGP preparations at intermediate steps of either Method of AGP purification: Affi-Blue FT, Cibacron Blue dye column flow-through, CHT, ceramic hydroxyapatite peak; HT Affi-Blue, HiTrap Cibacron Blue dye column flow-through; SSA Sup., supernatant following sulphosalicylic acid precipitation and neutralization. (c) depicts nitrocellulose blots of replicated gels containing 1.0 μg of purified human serum albumin (HSA) or of AGP purified by either Method 3 (M3) or Method 4 (M4) and probed either with Anti-AGP antibodies (at left) or Sambucus nigra agglutinin (SNA lectin) at left. The position of molecular mass markers and their mass in kDa is shown at left of each panel.

Figure 4

In vivo clearance, circular dichroism spectra, and deglycosylation of AGP purified by Methods 3 or 4. (a) shows residual, normalized acid-precipitable radioactivity in rabbit plasma following injection of radioiodinated AGP purified as indicated by arrowed text. (b) depicts circular dichroism spectra of AGP purified by Method 3 (upper) and Method 4 (lower); Δε represents the difference between the absorption by AGP of left-handed and right-handed circularly polarized light at different wavelengths. (c) shows samples of AGP purified by either method, with (+) or without (−) reaction with PNGase F; reaction of commercial AGP (Sigma) is also shown. Molecular mass markers are shown at left in kDa.