Abstract
Guanase from human liver was purified by two methods.
The first was a procedure involving ammonium sulfate fraction, TEAE column chromatography, hydroxyapatite column chromatography, affinity chromatography on CM Affi-gel blue and gel filtration on Sephacryl S-200. Buffers containing 0.1mM/L of dithiothreitol were used in all steps. Dithiothreitol was added to cut the S-S complex of guanase if it was present. Guanase was purified 1505-fold with a 17% yield in this method. Homogeneity was established by SDS-polyacrylamide gel electrophoresis.
The second method was employed to purify guanase without using dithiothreitol. It involved four repetitions of ammonium sulfate fractionation together with TEAE chromatography and gel filtration. Guanase in the seccnd method was resolved into two fractions, guanase 1 and 2 on the first TEAE chromatography.
The molecular weight of the enzyme purified with dithiothreitol according to SDS-polyacrylamide gel electrophoresis and gel filtration was estimated to be 55000 and 64000, respectively. When dithiothreitol was not used, the molecular weight of guanase 1 was 64000 and that of guanase 2 was 110000 according to gel filtration. In SDS-polyacrylamide gel electrophoresis, the molecular weights of both guanase 1 and 2 were 55000. This shows that the enzyme is composed of a subunit with a molecular weight of 55000. These results indicate that guanase 2 consists of polymeric form.
As guanase from crude extract of human liver showed the same bands of guanase 1 and 2 on agarose gel, polymeric forms of guanase are considered toThe present in human liver.
