Follow-up enzyme assay

Enzyme stability was determined by incubating enzyme at various temperatures followed by enzyme assay. Results shown in Table 2 revealed that the enzyme was stable for up to one hour at 60°C, which therefore allowed a heat treatment step to be included in the purification scheme. 

DNA studies aim to provide ultimate proof of a disease in a patient with an inherited metabolic disorder and should not usually lead to follow-up assays. Indeed, enzyme assays and other functional studies are often easier, cheaper and have a higher sensitivity than molecular studies. When possible, enzyme assays should be performed prior to the DNA test. As always exceptions prove the rule. 

Enzyme assays were conducted in a 10 mL screw-neck glass test tube containing 100 fiL of lysate, 90 fiL of a 250 /ng/mL solution of 6-mercaptopurine in 0.01 M HC1, and 15 /uL of 250 mM sodium phosphate buffer (pH 9.2). Reactions were initiated by the addition of 32 fiL of a 3 1 mixture of 250 fiM S-adenosyl-L-methionine and 30 mM dithiothreitol. The final pH was 7.5. After a 1-hour incubation at 37°C, the reaction was stopped by the addition of 850 fjL of ice-cold 3.5 mM dithiothreitol and 50 fih of 1.5 M H2S04. The tubes were then heated at 100°C for 2 hours. To each tube, 500 fiL of 3.4 M NaOH was added, immediately followed by 8 mL of toluene-amyl alcohol-phenyl mercuric acetate. The tubes were shaken for 10 minutes and centrifuged. Then 6 mL of the toluene layer was transferred to a glass-stoppered conical test tube and 0.2 mL of 0.1 M HC1 added. After vortex-mixing and centrifuging, the toluene layer was discarded. Samples (50 fiL in 0.1 M HC1) were used for HPLC analysis. Product formation was linear for up to 120 minutes and 150 /u,L of lysate. 

Sohm et al. (in press) have extended their studies of APA and P uptake kinetics by Trichodesmium colonies in the N. Atlantic to a comparative study of these two indices in the tropical N. Pacific and coastal northern Australian waters as well. They detected sharp contrasts among the sites with much higher Riax and APA values in their tropical N. Atlantic stations compared to the N. Pacific and northern AustraHa indicating more severe P hmitation in the tropical N. Atlantic. Dyhrman et al. (2002) also reported evidence of severe P stress in Trichodesmium in the N. Atlantic based on a enzyme linked fluorescence (ELF) assay. A follow up study also saw less evidence of P stress in samples from the N. Pacific (Hynes et al., Pers. Comm.). 

In order to monitor SOD activity routinely, assays that require only instrumentation typical of a chemical or biochemical laboratory were set up. The assays consist of a reaction mixture that generates superoxide anion and a coupled redox reaction that scavenges the superoxide ion. The latter reaction is usually followed spectrophoto-metrically. The addition of superoxide dismutase destroys superoxide and inhibits the coupled reaction. The specific activity of the enzyme is determined on the basis of the amount of protein required to slow down to 50% the first-order coupled reaction, i.e., one enzymatic unit is the amount of protein that reduces the rate to 50% and the specific activity corresponds to the number of units per milligram of protein. 

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