Sulfhydryl Oxidase in Human Milk: Stability of Milk Enzymes in the Gastrointestinal Tract

Abstract

SummarySulfhydryl oxidase (SOX) is present in human milk and in milk from all species that have been studied. The pH optimum of human milk SOX is in the neutral range between 7.0 and 7.5. Human milk SOX is stable in an acid environment: 50% of its activity remains after 1 h at pH 2.5. Acid stability is also characteristic of 7-glutamyltranspeptidase, another membranebound enzyme in skim milk. SOX is resistant to pepsin (4000 U/ ml), trypsin (50 pg/ml), chymotrypsin (200 pg/ml), and to trypsin plus chymotrypsin (25 pg eachlml). Milk SOX activity has been detected in the stomach and proximal small intestine contents of suckling rats. Human and bovine SOX are relatively heat stable: 75% of the latter remains after treatment at 62.S°C for 30 min and 65% of the former remains after treatment at 60°C for 10 min. Neither remains after 62.5"C for 30 min. AbbreviationsDTT, dithiothreitol GGT, 7-glutamyltranspeptidase GI, gastrointestinal GSH, reduced glutathione GSSG, oxidized glutathione SIgA, secretory IgA SOX, sulfhydryl oxidase Some 60-70 enzymes have been reported to be present in milk (4, 35) but little is known about their function. Milk enzymes may act in the milk itself, in the intestine, or may be absorbed by intestinal epithelial cells and transported to target tissues. At least two human milk enzymes, bile salt-stimulated lipase and mammary amylase, have been shown to play a physiologic role in the digestion of milk nutrients (15,18,19,22,23,28). It is unlikely, considering the strict control of secretory processes (29), that a significant number of enzymes occur in milk simply because of "spillage" from the blood or acinar cells.Sulfhydryl oxidases are a class of enzymes that catalyze the net synthesis of disulfide bonds (21). These enzymes use molecular oxygen and both low molecular weight sulfhydryl compounds and protein sulfhydryls as substrates in vitro (3, 3 1, 34); they produce H202 plus a disulfide. Sulfhydryl oxidases were originally described in bovine milk (24) but have subsequently been found in skin (38), seminal vesicle (31), epididymis (6), and in mouse plasmacytomas producing IgM (34). The in vivo substrates for SOX in mouse plasmacytomas were shown to be IgM monomers and the associated J chain (34). But in vivo substrates for the sulfhydryl oxidases found in mammalian secretions and tissues have not been established. Milk SOX might play a role in altering the structure of milk or intestinal proteins that are dependent upon the redox state of their sulfhydryls for function.The present experiments were designed to determine whether or not SOX and other milk enzymes had stability characteristics similar to those of milk proteins, such as secretory immunoglobulins (13, 25), which have been shown to be active in the gastrointestinal tract of the suckling neonate. Studies, both in vitro and in vivo, were done in order to measure the stability of SOX to acid and to gut protease. MATERIALS AND METHODSHuman milk samples were obtained through the La Leche League of Staten Island. The milk was frozen im...

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