1. A study has been made of the enzymic hydroxylation of biphenyl by liver microsomal preparations from 11 species of animals, by using a fluorescence method for the micro-estimation of the hydroxylation products, 2- and 4-hydroxybiphenyl. 2. Livers from all species examined produced 4-hydroxybiphenyl, but only those from mice, hamsters, cats, coypus and frogs produced 2-hydroxybiphenyl as well. 3. Adult rat and rabbit livers produced only the 4-isomer, but livers from the young of these species also produced the 2-isomer. 4. The properties and requirements of the 4-hydroxylating enzyme of rabbit liver were studied. 5. The results are discussed and it is suggested that the 2- and 4-hydroxylating enzymes are different.

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH(2) and oxygen. Activity with NADH(2) was one-third of that with NADPH(2). 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at -12 degrees . Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2'-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.

1. The variation with pH of the excitation and fluorescence wavelengths and fluorescence intensity of several hydroxy- and amino-biphenyls has been investigated with a view to using the data for the determination of hydroxybiphenyls in biological material. 2. 2- and 3-Hydroxy-, 2,2'-dihydroxy-, and 2-, 3- and 4-amino-biphenyl exhibited excited-state ionization. 4-Hydroxy- and 4,4'-dihydroxybiphenyl did not, and showed normal fluorescence changes with pH. 3. Biphenyl and 2- and 4-methoxybiphenyl showed no changes in fluorescence in the range pH0-14. 4. 4,4'-Dihydroxybiphenyl showed fluorescent peaks corresponding to the un-ionized form, the monoanion and dianion at the appropriate pH values, but 2,2'-dihydroxybiphenyl showed fluorescent peaks corresponding to the un-ionized form and the monoanion only, suggesting that it did not form a dianion. 5. 3- and 4-Nitrobiphenyl were non-fluorescent. 6. These results are discussed.

No abstract

No abstract