There are many reports of enzymatic determination of glycerol, but there are basically three enzymes. That is, glycerol oxidase, glycerol inactivating enzyme, glycerol dehydrogenase.
- Oxidase method
If glycerol is decomposed by glycerol oxidase, it is converted to glyceraldehyde and hydrogen peroxide. The hydrogen peroxide was synthesized by peroxidase and 4-aminoantipyridyl carbamate and phenol to form red compounds. The spectroscopic analysis was carried out at 546 nm. In addition, the oxygen consumed can also be determined directly with an oxygen electrode.
- Activating Enzyme Method
In the presence of ATP (adenosine triphosphate), glycerol is decomposed by glycerol-inactivating enzymes and converted to glycerol phosphoric acid and ADP (adenosine diphosphate).
glycerol + ATP →Glycerol-3-phosphoric acid + ADP
Glycerol was quantitatively determined by the following enzymatic reactions with glycerol-3-phosphate and adenosine diphosphate.
In the presence of oxygen, when glycerol-3-phosphoric acid is used, glycerol-3-phosphoric acid reacts with oxidase and converts to dihydroxyketone phosphoric acid and hydrogen peroxide. At this time, the content of glycerol can be quantitatively determined by directly measuring the consumption of oxygen or by using oxygen electrode to determine the consumption of oxygen.
In addition, hydrogen peroxide can be quantitatively determined by spectroscopic analysis at 546 nm, using peroxidase and 4-aminoantipyridine and phenol to form red compounds.
In addition, glycerol-3-phosphate and NAD [coenzyme A (pyridine diphosphate nucleotide)] were transformed into dihydroxy ketone phosphate and NADH (reductive coenzyme A) by glycerol phosphorus dehydrogenase. The NADH was analyzed at 340 nm, and the content of glycerol could be quantitatively determined.
Glycerol-3-phosphoric acid +NAD →Dihydroxy ketone phosphoric acid+NADH
When ADP (adenosine diphosphate) is used, phosphoenol group and ADP are converted to pyruvate and A TP (adenosine triphosphate), then pyruvate and NAD (coenzyme A) are reacted to convert pyruvate (lactic acid) dehydrogenase to pyruvate and NADH (reduced coenzyme A).
ADP+Phosphoenolenol→ Pyruvic acid+ATP
Pyruvic acid+NAD→pyruvate+NADH
The determination of NADH can quantitatively determine glycerol.
- Dehydrogenase Method
The glycerol is decomposed by glycerol dehydrogenase and converted into dihydroxy ketone and NADH.
Glycerol+NAD(CoA)Dihydroxyketone+NADH
The reaction of NADH or nitrotetrazolium salt, one of the tetrazolium salts, with NADH was directly determined to form L-4-acetyl-5-aminomethyl pyrrole. The spectra were analyzed at 546 nm.
According to this fermentation method, triglycerides can be quantitatively determined. That is to say, glycerol and fatty acid are converted by lipase or esterase, and the content of glycerol ester is calculated by quantitative determination of glycerol. This method has been used in blood tests and so on.
