CN109085269B - Method for studying reaction behavior of PQQ with Lys and Arg - Google Patents
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Abstract
A method for researching reaction behaviors of PQQ, Lys and Arg is the first part of the research on inhibition of PQQ and advanced glycosylation end products AGEs, and belongs to the technical field of bioanalysis. Lys and Arg are the major free amino acids of DNA involved in the glucose reaction. In order to clarify the action mechanism, the invention researches the reaction behavior of PQQ, Lys and Arg, optimizes the UPLC chromatographic separation condition, avoids the damage of a chromatographic column caused by using a high-concentration salt solution, and separates the enantiomer. The method determines a standard curve, accuracy and detection limit of the concentration of PQQ, determines the concentration of free PQQ reacted with Arg and Lys, identifies the structure of a reaction product of PQQ and Lys or Arg by UPLC-MS, finds a new compound PQQ-Lys and PQQ-Arg by determining a mass spectrum result, also finds that a molecule of PQQ can react with a molecule of Lys and a molecule of Arg simultaneously to obtain a new compound PQQ-Lys-Arg, inhibits the formation of AGEs by the reaction of PQQ and Lys and Arg, and provides an experimental basis for discussing a possible mechanism for inhibiting the formation of AGEs by PQQ.
Description
Technical Field
A method for researching the reaction behavior of PQQ (pyrroloquinoline quinone) with Lys (lysine) and Arg (arginine) is the first part of the research on the inhibition effect of PQQ and advanced glycosylation end products AGEs, and belongs to the technical field of bioanalysis.
Background
Advanced glycation end products (AGEs) are chronic, non-enzymatic glycosylation products of proteins. AGEs are ubiquitous in normal humans, and plasma AGEs levels increase with age, which is an important biomarker of aging of the body, and AGEs levels are generally increased in diabetic patients and the elderly. It has been reported that the formation of AGEs may occur during the formation of plaques in early Alzheimer's Disease (AD). AGEs bind to cell surface RAGE (AGEs receptor) and can cause oxidative stress, trigger an inflammatory cascade, atherosclerosis, and even thrombosis. The accumulation of AGEs in the body causes various complications, and thus AGEs is a worthy target to be studied.
The formation of AGEs involves a complex series of reactions, first, carbonyl groups of sugars and free amino acids (mainly lysine Lys and arginine Arg) of protein DNA are covalently bound to form schiff (Schiffbase) bases, which are unstable and rapidly rearrange to obtain more stable Amadori products (early glycosylation products), and then slowly rearrange and dehydrogenate the Amadori products to obtain AGEs (see reaction formula 1). AGEs can cause endothelial cell apoptosis and increase the production of Reactive Oxygen Species (ROS), leading to altered mitochondrial function, and these factors are highly correlated with the pathogenesis of diabetes, atherosclerosis, tumors, aging, and degenerative diseases.
Prevention of these diseases by inhibiting the accumulation of AGEs in the body is therefore a very promising potential approach. It is hypothesized that when a reducing sugar is covalently bonded to a protein DNA free amino acid (Lys or Arg) amino group to form a Schiff base in the first step of AGEs formation reaction shown in equation 1, the Schiffbase base formed by the reaction with the reducing sugar can be prevented by competing with the Lys or Arg amino group with an inhibitor, and ultimately the formation of AGEs can be inhibited.
More and more studies have shown that foods rich in polyphenols have a strong protective effect on the formation of AGEs. Pyrroloquinoline quinone (PQQ) and the polyphenols have similar biological factors and have strong protective effects on neurodegenerative disease cognitive impairment caused by oxidative stress.
PQQ is an oxidoreductase prosthetic group, which is mainly distributed in prokaryotes and parts of plants and mammals. PQQ has strong antioxidant ability, and can improve cognitive function impairment caused by oxidative damage and oxidative stress in neurodegenerative diseases such as aging; can inhibit the accumulation of amyloid protein and has prospect in the aspect of treating nervous system diseases. In addition, it has recently been reported that oral administration of PQQ improves impaired glucose tolerance in type 2 diabetic mice and reduces oxidative damage in different organs in diabetic mice.
PQQ has a special structure and various special chemical properties, and is easily combined with various substances such as amino acids, neurotransmitters and the like. In research, we found that conjugated quinone bonds in PQQ can be converted into useful derivatives through amino group binding of amino acids, thereby acting as free radical scavenging. However, the mechanism of action is not fully understood. We have studied the reaction of PQQ with neurotransmitters such as glycine and glutamic acid by HPLC, and separated PQQ from the reaction product using an ion-pair reagent and a buffer salt solution of a certain concentration as a mobile phase. The literature reports that only PQQ forms oxazole (oxazole) compounds with one molecule of amino acid. Lys and Arg are main free amino acids of DNA involved in glucose reaction, whether PQQ can react with Lys and Arg, whether PQQ has the function of inhibiting AGEs, and whether the antioxidant capacity of PQQ is related to the formation of AGEs; in order to further clarify the mechanism of action, the present inventors studied the reaction behavior of PQQ with Lys and Arg, and found novel compounds PQQ-Lys, PQQ-Arg, and PQQ-Lys-Arg (reaction formula 2) as a result of mass spectrometry, thereby providing experimental evidence for studying the possible mechanism of inhibiting AGEs formation by PQQ.
Equation 2 PQQ is reacted with arginine and lysine.
Disclosure of Invention
The purpose of the present invention is to provide a method for studying the reaction behavior of PQQ (pyrroloquinoline quinone) with Lys (lysine) and Arg (arginine), which are the main free amino acids of DNA involved in the glucose reaction. In order to further clarify the mechanism of action, the present inventors studied the reaction behavior of PQQ with Lys and Arg, and found novel compounds PQQ-Lys, PQQ-Arg, and QQ-Lys-Arg (equation 2) as a result of mass spectrometry, thereby providing experimental evidence for studying the possible mechanism of inhibiting AGEs formation by PQQ.
The invention adopts ACQUITY UPLC @ BEH C18 column (2.1X 100mm,1.7 μm), formic acid is added into a mobile phase, gradient elution is carried out with a methanol system, and the reaction products of PQQ, Lys and Arg are separated. The novel compounds PQQ-Lys and PQQ-Arg were discovered by UPLC-MS, and it was also discovered that one molecule of PQQ can react with one molecule of Lys and one molecule of Arg at the same time to give the novel compound PQQ-Lys-Arg, and the enantiomers are also isolated. Avoids the damage of the chromatographic column caused by using high-concentration salt solution.
The technical scheme of the invention is as follows: the method for researching the reaction behavior of PQQ with Lys and Arg comprises the following steps:
1. UPLC chromatographic separation conditions
UPLC analysis conditions, column:ACQUITY UPLC @ BEH C18 column (2.1X 100mm,1.7 μm), mobile phase A: h2O +0.1% formic acid, B: methanol; 2/98B/A was eluted for 5min, with a gradient of 20min to 60/40B/A. PDA detection (photo-electric secondary array tube detector). After 0.1% formic acid is added to the mobile phase and gradient elution is performed, the PQQ can be better separated from the reaction product.
2. Standard Curve, accuracy and detection Limit determination
Accurately weighing a certain amount of PQQ, preparing standard solutions with the concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0mM, injecting samples respectively, drawing the obtained integral area to the concentration of the sample, and performing linear regression to obtain a regression equation of y =6E +06x-7834.9, wherein R is2= 0.9998; linear range of 0.2-1.0 mM; as shown in fig. 1.
The sample was continuously injected 6 times on the same day under the above chromatographic conditions, and the relative standard deviation was 2.2% (n =6) on every three consecutive days (4.3% (n =3) on the day, calculated from the peak area. The minimum detection amount is 2.0ng (S/N is more than or equal to 3);
measurement of free PQQ concentration after reaction of PQQ with Arg and Lys
PQQ was mixed with Arg and Lys at a v/v ratio of 1/1, PQQ was mixed with Arg and Lys at a v/v ratio of 1/1/1, incubated in a 37 ℃ water bath under dark conditions, 10. mu.L of the reaction solution was extracted at each time point of 0, 0.5, 1, 2, 4, 6, 8 and 24 hours, UPLC analysis was performed, and the peak area obtained was substituted into a linear equation to calculate the concentration of free PQQ. The resulting PQQ concentration was plotted against time to obtain a graph of PQQ versus reaction time (fig. 2). The reaction speed of PQQ and Lys is faster than that of PQQ and Arg, and the reaction of PQQ and Lys is complete after 24 h. The reaction rate of PQQ and Arg is lower than that of PQQ and Lys, and the reaction is basically stable after 8h, so that PQQ can not be completely reacted. When Arg and Lys are simultaneously present, PQQ rapidly and completely reacts.
4. Structure for identifying reaction product of PQQ and Lys or Arg by UPLC-MS
The UPLC chromatographic conditions are the same as the above, and the mass spectrum Detector SQ Detector 2 and the PDA Detector optimize mass spectrum parameters: desolventizing gas temperature: 400 ℃; desolventizing agent gas flow: 600L/h; ion source temperature: 110 ℃; taper hole gas flow: 50L/h; capillary voltage: 3000V; taper hole voltage: 30V; mass spectrometry was performed using electrospray positive ion mode ESI +.
In the ESI-MS spectrum (FIG. 3) of the reaction solution of PQQ and Arg, the obtained ion was M/z 441.32, which is the M +1 molecular ion peak of PQQ-Arg with a molecular weight of 440. There was a 331, PQQ M +1 peak at the same time. The chromatogram showed a PQQ chromatogram peak with a molecular weight of 330 (retention time 3.29 min) in addition to the PQQ-Arg product peak of 440 (retention time 1.55min,2.23min, respectively, as enantiomers).
The ESI-MS spectrum (FIG. 4) of the reaction solution of PQQ and Lys showed that the obtained ion was M/z 413.27(M +1), 414.20(M +2), and it was a molecular ion peak of PQQ-Lys having a molecular weight of 412. The chromatogram showed the presence of the product in enantiomers (retention times 1.75min and 2.72 min).
When PQQ is present together with Arg and Lys, one molecule of PQQ binds to one molecule of Arg and one molecule of Lys, respectively, and the resultant ion in ESI-MS spectrum (FIG. 5) of the reaction solution is M/z 525.32(M +1) and 526.69(M +2), which are molecular ion peaks of PQQ-Arg-Lys with a molecular weight of 524. The chromatogram showed the presence of the enantiomers (retention times of 2.22min and 2.76 min).
The invention has the beneficial effects that: the invention provides a method for researching the reaction behavior of PQQ (pyrroloquinoline quinone) with Lys (lysine) and Arg (arginine), wherein the Lys and the Arg are main free amino acids of DNA participating in glucose reaction. In order to further clarify the mechanism of action, the present inventors studied the reaction behavior of PQQ with Lys and Arg, and found novel compounds PQQ-Lys, PQQ-Arg, and QQ-Lys-Arg (equation 2) as a result of mass spectrometry, thereby providing experimental evidence for studying the possible mechanism of inhibiting AGEs formation by PQQ.
The invention adopts ACQUITY UPLC @ BEH C18 column (2.1X 100mm,1.7 μm), formic acid is added into a mobile phase, gradient elution is carried out with a methanol system, and the reaction products of PQQ, Lys and Arg are separated. The UPLC-MS is used to find new compounds PQQ-Lys and PQQ-Arg, and also finds that one molecule of PQQ can simultaneously react with one molecule of Lys and one molecule of Arg to obtain a new compound PQQ-Lys-Arg, and enantiomers are also separated. Avoids the damage of the chromatographic column caused by using high-concentration salt solution.
Drawings
FIG. 1 is a plot of integrated area versus standard PQQ concentration.
FIG. 2 is a graph of PQQ concentration versus reaction time.
FIG. 3 is a chromatogram and a mass spectrum of a reaction product of PQQ and Arg.
FIG. 4 is a chromatogram and mass spectrum of a reaction product of PQQ and Lys.
FIG. 5 is a chromatogram and mass spectrum of the reaction product of PQQ and Arg \ Lys.
Detailed Description
Example 1
1. UPLC chromatographic separation conditions
UPLC analysis conditions, column: ACQUITY UPLC @ BEH C18 column (2.1X 100mm,1.7 μm), mobile phase A: h2O +0.1% formic acid, B: methanol; 2/98B/A was eluted for 5min, with a gradient of 20min to 60/40B/A. And (6) PDA detection. After 0.1% formic acid is added to the mobile phase and gradient elution is performed, the PQQ can be better separated from the reaction product.
2. Standard Curve, accuracy and detection Limit determination
Accurately weighing a certain amount of PQQ, preparing standard solutions with the concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0mM, injecting samples respectively, drawing the obtained integral area to the concentration of the sample, and performing linear regression to obtain a regression equation of y =6E +06x-7834.9, wherein R is2= 0.9998; linear range of 0.2-1.0 mM; as shown in fig. 1.
The sample was continuously injected 6 times on the same day under the above chromatographic conditions, and the relative standard deviation was 2.2% (n =6) on every three consecutive days (4.3% (n =3) on the day, calculated from the peak area. The minimum detection amount is 2.0ng (S/N is more than or equal to 3);
measurement of free PQQ concentration after reaction of PQQ with Arg and Lys
Mixing PQQ with Arg and Lys at a v/v ratio of 1/1, mixing PQQ with Arg and Lys at a v/v ratio of 1/1/1, incubating in a water bath at 37 ℃ in the absence of light, extracting 10 μ L of reaction solution at each time point of 0, 0.5, 1, 2, 4, 6, 8 and 24h, analyzing by UPLC, substituting the obtained peak area into a linear equation, and calculating the concentration of free PQQ. The resulting PQQ concentration was plotted against time to obtain a graph of PQQ versus reaction time (fig. 2). The reaction speed of PQQ and Lys is faster than that of Arg, and the reaction of PQQ and Lys is complete after 24 h. The reaction speed of PQQ and Arg is lower than that of Lys, the reaction basically tends to be stable after 8h, and PQQ can not be completely reacted. When Arg and Lys are simultaneously present, PQQ rapidly and completely reacts.
4. Structure for identifying reaction product of PQQ and Lys or Arg by UPLC-MS
The UPLC chromatographic conditions are the same as the above, and the mass spectrum Detector SQ Detector 2 and the PDA Detector optimize mass spectrum parameters: desolventizing gas temperature: 400 ℃; desolventizing agent gas flow: 600L/h; ion source temperature: 110 ℃; taper hole gas flow: 50L/h; capillary voltage: 3000V; taper hole voltage: 30V; mass spectrometry was performed using electrospray positive ion mode ESI +.
In the ESI-MS spectrum (FIG. 3) of the reaction solution of PQQ and Arg, the obtained ion was M/z 441.32, which is the M +1 molecular ion peak of PQQ-Arg with a molecular weight of 440. There was a 331, PQQ M +1 peak at the same time. The chromatogram showed a PQQ chromatogram peak with a molecular weight of 330 (retention time 3.29 min) in addition to the PQQ-Arg product peak of 440 (retention time 1.55min,2.23min, respectively, as enantiomers).
The ESI-MS spectrum (FIG. 4) of the reaction solution of PQQ and Lys showed that the obtained ion was M/z 413.27(M +1), 414.20(M +2), and it was a molecular ion peak of PQQ-Lys having a molecular weight of 412. The chromatogram showed the presence of the enantiomer of the product (retention time tr1.75min,2.72 min).
When PQQ is present together with Arg and Lys, one molecule of PQQ binds to one molecule of Arg and one molecule of Lys, respectively, and the resultant ion in ESI-MS spectrum (FIG. 5) of the reaction solution is M/z 525.32(M +1) and 526.69(M +2), which are molecular ion peaks of PQQ-Arg-Lys with a molecular weight of 524. The chromatogram showed the presence of the enantiomers (retention times of 2.22min and 2.76 min).
Claims (1)
- A method for researching the reaction behavior of PQQ with Lys and Arg, which is characterized by comprising the following steps:(1) UPLC chromatographic separation conditionsUPLC analysis conditions, column: ACQUITY UPLC @ BEH C18 column 2.1X 100mm,1.7 μm, mobile phase A: h2O +0.1% formic acid, B: methanol; 2/98B/A for 5min, and eluting with gradient of 20min to 60/40B/A; detecting by a PDA; adding into mobile phaseAfter 0.1 percent formic acid and gradient elution, PQQ can be well separated from a reaction product;(2) standard Curve, accuracy and detection Limit determinationAccurately weighing a certain amount of PQQ, preparing standard solutions with the concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0mM, injecting samples respectively, drawing the obtained integral area to the concentration of the sample, and performing linear regression to obtain a regression equation of y =6E +06x-7834.9, wherein R is2= 0.9998; linear range of 0.2-1.0 mM;continuously sampling for 6 times in the same day under the chromatographic condition, and calculating according to peak area to obtain a relative standard deviation of 2.2% and n =6 in the day; continuously injecting samples for three days every other day, wherein the standard deviation in the daytime is 4.3%, and n = 3; the minimum detected quantity is 2.0ng, and S/N is not less than 3;(3) measurement of free PQQ concentration after reaction of PQQ with Arg and LysMixing PQQ with Arg and Lys respectively according to a v/v ratio of 1/1, mixing PQQ with Arg and Lys according to a v/v/v ratio of 1/1/1, carrying out light-shielding reaction, placing in a water bath at 37 ℃ for incubation, extracting 10 mu L of reaction liquid at each time point of 0, 0.5, 1, 2, 4, 6, 8 and 24h, carrying out UPLC analysis, substituting the obtained peak area into a linear equation, and calculating the concentration of free PQQ; the obtained PQQ concentration is plotted against time to obtain a PQQ-reaction time graph, the reaction speed of PQQ and Lys is faster than that of PQQ and Arg, and the reaction of PQQ and Lys is complete after 24 h; the reaction speed of PQQ and Arg is lower than that of PQQ and Lys, the reaction basically tends to be stable after 8 hours, and PQQ can not be completely reacted; in the case of Arg and Lys, PQQ reacts rapidly and completely;(4) structure for identifying reaction product of PQQ and Lys or Arg by UPLC-MSThe UPLC chromatographic conditions are the same as the above, and the mass spectrum Detector SQ Detector 2 and the PDA Detector optimize mass spectrum parameters: desolventizing gas temperature: 400 ℃; desolventizing agent gas flow: 600L/h; ion source temperature: 110 ℃; taper hole gas flow: 50L/h; capillary voltage: 3000V; taper hole voltage: 30V; mass spectrum adopts ESI + of electrospray positive ion mode;in ESI-MS spectrum of reaction solution of PQQ and Arg, the obtained ion is M/z 441.32, which is M +1 molecular ion peak of PQQ-Arg with molecular weight 440; the 331, PQQ M +1 peak was present at the same time; the chromatogram has a PQQ-Arg product peak with molecular weight 440, retention time of 1.55min and 2.23min, and a PQQ chromatogram peak with enantiomeric molecular weight 330, retention time of 3.29 min;in ESI-MS spectrogram of PQQ and Lys reaction solution, the obtained M/z M +1 ion peak is 413.27, M +2 ion peak is 414.20, and the peak is molecular ion peak of PQQ-Lys with molecular weight of 412; the chromatogram showed the presence of the enantiomer: the retention time is 1.75min and 2.72 min;when PQQ exists together with Arg and Lys, one molecule of PQQ is combined with one molecule of Arg and one molecule of Lys respectively, and in an ESI-MS spectrogram of a reaction solution, an M/z M +1 ion peak is 525.32, an M +2 ion peak is 526.69, the peak is a molecular ion peak of PQQ-Arg-Lys with a molecular weight of 524, and the chromatogram shows that an enantiomer exists and retention time is 2.22min and 2.76 min.
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