BACTERICIDAL DOMAIN OF LACTOFERRIN - DETECTION, QUANTITATION, AND CHARACTERIZATION OF LACTOFERRICIN IN SERUM BY SELDI AFFINITY MASS-SPECTROMETRY

Lactoferricin is a bioactive peptide fragment (3196 Ha) derived from l actoferrin (80 kDa) that contains the bactericidal domain and the lymp hocyte receptor-binding domain of lactoferrin. Although lactoferricin has: been produced from lactoferrin by proteolytic digestion in vitro, its natural occurrence and distribution in vivo are still not clear, in part because of the absence of a suitable detection means. Surface- enhanced laser desorption/ionization (SELDI) was used to detect and ch aracterize lactoferricin by affinity mass spectrometry. Human, porcine , and bovine lactoferricin in unfractionated serum samples were found to bind specifically to ligands presenting a terminal n-butyl group. S ELDI was used to detect and quantify each species of lactoferricin in a manner that was independent of the presence of intact lactoferrin, p artially degraded lactoferrin, and lactoferrin peptides containing the lactoferricin peptide sequence. The limit of detection of bovine lact oferricin in serum was as low as 200 pg/ml. The FKCRRWQWR-homoserine/- homoserine lactone moiety of bovine lactoferricin, which includes the complete antimicrobial center (i.e., RRWQWR), was shown to be responsi ble for interaction with the n-butyl group. The SELDI procedure define d here is the only molecular recognition tool known to date that is ca pable of distinguishing the multi-functional lactoferricin domain loca ted within structurally related but distinct forms of lactoferrin and its metabolic fragments. Enabling the direct quantitation of lactoferr icin produced in vivo opens new opportunities to evaluate lactoferrin function. (C) 1998 Academic Press.