FIGURE 5.
Analysis of tetrameric and monomeric β-tryptase associated with G4 Fab by Superose 12 gel filtration chromatography. Absorbance at 280 nm was continuously monitored, and elution times were recorded at each protein peak. Total elution time was 20 min. A, β-Tryptase tetramers (1 μg) stabilized with 1.5 μg of heparin were incubated with 6.7 μg of G4 Fab (4-fold molar excess) in 250 μl of PBS (pH 7.4), at room temperature for 1 h and applied to a Superose 12 column equilibrated with 10 mM HEPES running buffer (pH 7.4), containing 1 M NaCl. B, β-Tryptase monomers (1 μg) incubated with 6.7 μg of G4 Fab were subjected to Superose 12 chromatography as described. C, Elution pattern of G4 Fab (6.7 μg) showed a retention time of 14.6 min. D, Elution pattern of β-tryptase tetramers (1 μg) showed a retention time of 11.9 min. E, Elution pattern of β-tryptase monomers (1 μg) showed a retention time of 14.8 min. Peak OD280 values for each major peak were 4.4 × 10−3, 4.9 × 10−3, 5.7 × 10−3, 1.4 × 10−3, and 0.4 × 10−3 (A–E, respectively. Retention times of various standards were as follows: thyroglobulin (8.9 min, 669 kDa), apoferritin (10.7 min, 443 kDa), β-amylase (11.6 min, 200 kDa), alcohol dehydrogenase (12.3 min, 150 kDa), BSA (13.0 min, 66 kDa), and carbonic anhydrase (14.7 min, 29 kDa).