Molecular weight distributions of α-lactalbumin polymers formed by mammalian and microbial transglutaminases

Polymer formation of α-lactalbumin (α-LA) caused by the incubation with transglutaminases from guinea pig liver (GTGase) and Streptoverticillium mobaraense (MTGase) in the presence of dithiothreitol (DTT) was analyzed by gel permeation chromatography combined with a multi-angle laser light scattering detector (GPC–MALLS) system using the elution buffer containing sodium dodecyl sulphate. Polymer formation by the two enzymes was carried out in the absence as well as in the presence of Ca 2+ , because we had found previously that not only Ca 2+ -independent MTGase but also Ca 2+ -dependent GTGase could cross-link α-LA in the absence of Ca 2+ . In the Ca 2+ -containing system, the incubation with TGases produced precipitates in 30 min, thereby inhibiting the growth of polymer. The maximum sizes of polymers were constant from 30 to 60 min, i.e. 3.0×10 6 and 7.0×10 6 , for GTGase-catalyzed and MTGase-catalyzed cases, respectively. On the contrary, the large polymers (>1×10 7 ) were formed by the incubation with the enzymes in the Ca 2+ -free system. These results suggest that TGases could not make full use of catalytic activity in the presence of Ca 2+ because of the difficult access of the enzymes to the targeting sites in aggregates formed via electrostatic interactions. Irrespective of the presence or absence of Ca 2+ in the reaction mixtures, GTGase showed the high cross-linking activity at the early stages of incubation, namely, the more rapid decrease of monomer α-LA and the more rapid growth of large polymers. In the late stages (prolonged incubation), however, reverse results were observed with respect to the efficiency in polymer formation, i.e. MTGase was capable of forming larger polymers than GTGase.