Identification of lactoperoxidase in mature human milk

Myeloperoxidase (MPO) derived from milk leukocytes and lactoperoxidase (LPO ) secreted from the mammary gland have been identified previously in human colostrum. These peroxidases are known to play host defensive roles through antimicrobial activity. The goals of this study were to measure the peroxi dase activity in mature human milk and to characterize the enzyme responsib le for the activity. As determined using 3,3',5,5'-tetramethylbenzidine as substrate, whey prepared from human milk samples obtained 1 and 5 months po stpartum showed levels of peroxidase activity equivalent to 0.13 +/- 0.18 a nd 0.24 +/- 0.21 mu g/mL bovine LPO (bLPO; n = 13), respectively. Whey from early milk was fractionated into two peaks of peroxidase activity by catio n-exchange chromatography; the peroxidase in the first peak was sensitive t o dapsone, which is an inhibitor of LPO, whereas the second peroxidase was not. Whey from mature milk showed only the first peak. Purified bLPO and MP O showed chromatographic behaviors that were similar to the first and secon d peaks, respectively. The dapsone-sensitive peroxidase from mature milk wa s further purified (952-fold from whey) by hydrophobic interaction chromato graphy. This preparation showed two bands with molecular masses of 80 and 9 0 kDa by polyacrylamide gel electrophoresis and immunoblotting using an ant ibody against bLPO. After deglycosylation, two distinct proteins with lower molecular weights were observed. Amino acid sequencing indicated that both of these proteins are LPO. These results provide evidence that LPO is pres ent in mature human milk and that it is responsible for most of the peroxid ase activity in mature milk. (J. Nutr. Biochem. 11:94-102, 2000) (C) Elsevi er Science Inc. 2000. All rights reserved.