Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models.
The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light scattering. The concentration (c) dependence of the apparent weight-average molecular weight (Mwapp) was determined over a wide concentration range of 0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme concentration, corresponded to the Mr value of the monomer. The self-association mode of the apoenzyme was systematically explored with nonlinear least-squares analysis of the Mwapp versus c data. The simplest model that fitted the data well was a model of isodesmic indefinite self-association of the monomer with the isodesmic association constant of 0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20 degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8, 3829-3838), did not fit the Mwapp versus c data either in the limited low concentration range or in the whole concentration range examined at 25 degrees C. To test the validity of the chosen model, the observed sedimentation boundary profiles were compared with the idealized boundary profiles calculated for the better-fit models. The profile calculated with the model of the isodesmic indefinite self-association mechanism was qualitatively consistent with the observed ones. The utility of the nonlinear least-squares procedure for analyzing self-associating systems was demonstrated.
Tojo
H
H
Horiike
K
K
Shiga
K
K
Nishina
Y
Y
Watari
H
H
Yamano
T
T
D000818:Animals; D001051:Apoenzymes; D001059:Apoproteins; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003605:D-Amino-Acid Oxidase; D011756:Diphosphates; D007668:Kidney / Q000201:enzymology*; D007834:Lasers; D046911:Macromolecular Substances; D008956:Models, Chemical; D008970:Molecular Weight; D012542:Scattering, Radiation; D012996:Solutions; D013552:Swine
D001051:Apoenzymes; D001059:Apoproteins; D011756:Diphosphates; D046911:Macromolecular Substances; D012996:Solutions; D003605:D-Amino-Acid Oxidase