A comparative study of l-lactablumins and of their relationship to lysozymes

Abstract

A study has been made of the occurrence, isolation and properties of a-lactalbumins from a number of species. These results have been considered in relation to the role of a-lactalbumins in the lactose synthetase system and their h omology with lysozymes . 1. The chromatographic fractionation of bovine whey proteins has been studied in detail to investigate the heterogeneity of a-lactalbumin and to isolate the major component in high purity. 2. A second a-lactalbumin genetic variant (A) has been isolated from the milk of Droughtmaster breeds of cattle and compared with the more common B variant . a - lactalbumin A differs from B by the substitution of glutamine for arginine. 3. Three minor proteins (SC1, SC2 and FC) occur together with each major bovine genetic variant . These minor proteins have been identified as Q-lactalbumins from their amino acid compositions and activity in lactose synthetase. They differ from each other in their electrophoretic mobilities and solubility in water . The two proteins of lower mobility at pH 7.5 (SC1 and SC2) are glycoproteins containing 12-15 per cent carbohydrate . SCl differs from SC2 by the presence of one residue of sialic acid per mole. FC is probably the same minor protein observed by other workers in zone electrophoresis of a-lactalbumin samples . It is not a glycoprotein and probably differs from the major a-lactalbumin by containing one less amide group. 4. Bovine a-Iactalbumln exhibits a bimodal pattern on elution from columns of DEAE-Sephadex or DEAE-cellulose at pH 6.3-7.8. The bimodality is not observed in zone electrophoresis, sedimentation or gel filtration. The relative size of each peak on rechromatography depends on previous treatment of the protein. 5. Using the knowledge gained in fractionation of bovine whey proteins, a-lactalbumins having high activity ln lactose synthetase with bovine "A protein" have been isolated from human, porcine, ovine and kangaroo milk. The following order of reactivities for the a-lactalbumins has been determined: bovine > human > porcine > kangaroo. A bimodal elution pattern resembling that of the bovine a-lactalbumins has been found for the human but not for the pig or kangaroo proteins . This property may be related to the function in the lactose synthetase system. A minor a-lactalbumin similar to the bovine FC protein appears to occur in porcine, human and ovine milks . Ovine whey does not contain proteins of similar properties to bovine SC1 and SC2. The lactose and lysozyme contents of milk from one subspecies of echidna (Toao multiacule atus) has been found to increase with time of lactation. The isolated lysozyme exhibits weak activity in lactose synthetase in the presence of bovine "A protein" and the pH dependence of the reaction closely parallels that of the "A protein" alone. The lysozyme has an isoelectric point of 12.0-12.5 and has approximately 1.3-1.5 times the specific lytic activity as hen egg lysozyme and similar activity to the human lysozyme, also isolated in this present work, A lysozyme from a second sub-species of echidna (T.a. aculeatus) has been shown to be inactive in the lactose synthetase reaction and has lower electrophoretic mobility at pH 5.3 than the lysozyme from T. a. multiaculeatus. Platypus milk contains traces of lactose and lysozyme activity but no protein of similar function to the a-lactalbumins from other mammals. The lysozyme from Toa. multiaculeatus has an amino acid composition i n termedi ate between the other lysozymes and a-lactalbumins isolated in this present study and cannot be simply classified in either group . There are also differences in optical rotatory dispersion in the region 205-350 nm of these proteins .