EFFECTS OF MICROHETEROGENEITY IN HEN EGG-WHITE LYSOZYME CRYSTALLIZATION

In earlier sodium dodecylsulfate-polyacylamide gel electrophoresis (SD S-PAGE) studies it has been found that commonly utilized commercial he n egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covale ntly bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two different ly charged monomers in comparable amounts. To explore the origin of th ese microheterogeneous contaminants, purified HEWL (PHEWL) has been ox idized with hydrogen peroxide (0.0026-0.88 M) at various pH levels bet ween 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) band s in SDS-PAGE gels shows that hydrogen peroxide at 0.88 M in acetate b uffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had in addition to one of the two monomer form s found in HEWL and PHEWL, three other differently charged monomer for ms, each of them representing about 25% of the preparation. SDS-PAGE a nalysis of OHEWL yielded two closely spaced dimer bands with M-r = 28 000 and 27 500. In addition, larger HEWL oligomers with M-r = 1.7 mill ion and 320 000 were detected by gel-filtration fast protein liquid ch romatography with multiangle laser light Buffer strip scattering detec tion. Non-dissociating PAGE in large pore size gels at pH 4.5 confirme d the presence of these large oligomers in HEWL and OHEWL. Increased m icroheterogeneity resulted in substantial effects on crystal growth an d nucleation rate. On addition of 10 mu g(-1) mg ml(-1) OHEWL to 32 mg ml(-1) HEWL crystallizing solutions, both the number and size of form ing crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL sol utions on addition of 0.03-0.3 M hydrogen peroxide. Repartitioning of the dimer during crystallization at various temperatures between 277 a nd 293 K was analyzed by SDS-PAGE. The crystals contained less than or equal to 25%(w/w) of the oligomers in the solution, with no apparent temperature dependence of the repartitioning.