Hp located in specific granules of neutrophils is more glycosylated than plasma-derived Hp. (A) Pooled subcellular fractions isolated from peripheral-blood neutrophils and purified plasma Hp were analyzed by Western blot analysis using rabbit anti-human Hp antibody. Western blots to the left demonstrate pooled subcellular fractions highly enriched in azurophil granule proteins (α-fraction), specific granule protein (β₁-fraction), gelatinase granule proteins (β₂-fractions), and secretory vesicles containing mainly plasma proteins (γ-fractions). These Western blots show that the β-chain of Hp, primarily present in β₁- and β₂-fractions, has a higher molecular weight (approximately 45-65 kDa) than the β-chain of plasma-derived Hp (39 kDa) primarily present in γ-fractions (secretory vesicles). Western blots to the right demonstrate that Hp β-chains from specific granules (pooled β-fractions) and plasma samples have an identical molecular weight of 30 kDa after complete N-deglycosylation by PNGase-F. (B) Western blot analysis of nonreduced pooled β-fractions and plasma samples, prepared from patients with the Hp 1-1 and Hp 2-1 phenotypes with the use of rabbit anti-human Hp antibody. These Western blots demonstrate that Hp dimer and Hp multimers contained in pooled subcellular β-fractions (ie, specific granules) have higher molecular weights than those contained in plasma.