Aldehyde sugar reductase determination.

: There are several questions about the reaction system for determining the activity of aldehyde glycase: how is the
1, 67mmol/l sodium potassium phosphate buffer configured? There are also people with 50mmol/l potassium phosphate buffer which is better.
2, the entire reflection system configuration to pay attention to what issues? Finally add DL-Glyceal or NADPH.
concentrations are used to draw the standard curves of 3 and NADPH? Please give me advice, thank you.
: When determining the activity of enzymes, sometimes due to insufficient purification of enzymes, resulting in the impact of results, in the detection of aldehyde glycase activity should pay attention to the following points:
(i) interference with other enzymes and substances
such as
tissues
plasma often contains NADH-cell pigment C reductase, it will interfere with the determination of various reductase. The most typical example of clinical enzyme assay is the interference of acetone acid in the blood with the determination of acetaminophen
transsiase
in the blood, because the reaction system contains a large amount of lactic acid
dehydrogenase
and NADH, can be used with acetone acid reaction consumption NADH, resulting in a decrease in absorbance at 340nm, if this NADH drop is also counted as ALT activity will cause errors.
Other interference with CK as determined by adenosine
kinases
(AK) in red blood cells, ADP is the CK substrate in CK's enzyme-coupled system, but it is also ak's substrate, and both enzyme reactions produce ATP, in tool enzymes (sugared) Kinase and 6-phosphate glucose dehydrogenase) both produce NADH caused by 340nm of increased absorbance, the result is that CK assay results are high, in order to avoid such interference, so in the reaction system to add AK inhibitors AMP and adenosine 5' phosphoric acid.
(ii) Contamination of tool enzymes
In currently used in
reagents
is mostly extracted from animal tissue or bacteria, inevitably contaminating other enzymes, if not paying attention to this problem, will cause incorrect results. What's more, some tool enzymes are mixed with assay enzymes, which in this case produce a high background. Therefore, the tool enzyme used must be very pure, and check the content of contaminated enzymes, such as the determination of the achlorine amino transfer enzyme used in the apple acid dehydrogenase containing AST, according to IFCC regulations should not exceed 0.005%.
(iii) non-enzyme reaction
Some substrates are unstable, without the role of enzymes can react on their own, such as ALP substrate phosphoric acid to nitrophenol-matched substrate solution, room temperature placed overnight, that is, self-water interpretation released to nitro yellow. Also, when measuring aldehyde shrinkase, its substrate aldehyde
compound
can react with NAD plus non-enzyme to produce a compound with a similar NADH absorption
spectral
, which brings difficulties to the determination.
(iv) Analysis of contamination of containers
e.g. improper flushing, analysis of containers and pipes mixed with a variety of substances, may affect enzyme activity, such as trace heavy metals can make enzyme infestion, residual
surfactants
may inhibit enzyme activity.
(v) Precipitation formation
When using optical method to monitor enzyme reactions, if there is precipitation formation or the sinking of particles in tissue homogeneity will cause a change in absorbance, resulting in error in the measurement results, which is common in low substrate solubility and high substrate concentration in the reaction system. It can be γ GGT using anti-glutamine on nitrobenzene as a substrate.
.