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\begin{document}

\title{Synergism Variation between Intracellular Glutathione, Phycocyanin and
SOD in Microalgae by Carbon Quantum Dot Fluorescence}



\author[1]{shuyu liu}%
\author[1]{Xiaoyu Li}%
\author[1]{* Zitong}%
\affil[1]{no affiliation}%


\vspace{-1em}



  
  \date{May 22, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
:Based on the use of CQDs as fluorescent probe and covalent coupling
method to detect biological molecules with amino groups, this method was
applied to the analysis and detection of metabolism of Microcystis
aeruginosa. The metabolic changes of carboxyl biomolecules in
Microcystis aeruginosa were analyzed by covalent coupling method,
including GSH, phycocyanin and SOD enzyme. The changes of GSH content in
Microcystis aeruginosa at different growth stages and the correlation
between phycocyanin, SOD and GSH were analyzed from one-dimensional,
two-dimensional and three-dimensional fluorescence. The content of
phycocyanin and SOD reached the maximum on the 65th day, and GSH was
more sensitive to the growth and metabolism of microalgae and began to
decrease on the 50th day. GSH plays an important role in reducing the
external oxidative damage of microalgae cells at each growth stage of
microalgae cells. Among them, GSH synthesis, GSH/GSSG mutual
transformation, synthesis of phytochelating peptides (PC), ASA-GSH cycle
and metabolism and other physiological activities in Microcystis
aeruginosa have interactions, which play a key role in maintaining the
antioxidant properties of microalgae and regulating redox-sensitive
signal transduction.%
\end{abstract}%



\sloppy


\textbf{Synergism Variation between Intracellular Glutathione,
Phycocyanin and SOD in Microalgae by Carbon Quantum Dot Fluorescence}

\textbf{Shuyu Liu*, Xiaoyu Li , Zitong}

School of Environment and Chemical Engineering, Shanghai University,
Shanghai, 201800, P. R. China

* Corresponding Author: Shuyu Liu

Email: \emph{liushuyu@shu. edu. cn}

Ph: +86-13501947933

Fax: +86-021-6613787

\textbf{Abstract} :Based on the use of CQDs as fluorescent probe and
covalent coupling method to detect biological molecules with amino
groups, this method was applied to the analysis and detection of
metabolism of Microcystis aeruginosa. The metabolic changes of carboxyl
biomolecules in Microcystis aeruginosa were analyzed by covalent
coupling method, including GSH, phycocyanin and SOD enzyme. The changes
of GSH content in Microcystis aeruginosa at different growth stages and
the correlation between phycocyanin, SOD and GSH were analyzed from
one-dimensional, two-dimensional and three-dimensional fluorescence. The
content of phycocyanin and SOD reached the maximum on the 65th day, and
GSH was more sensitive to the growth and metabolism of microalgae and
began to decrease on the 50th day. GSH plays an important role in
reducing the external oxidative damage of microalgae cells at each
growth stage of microalgae cells. Among them, GSH synthesis, GSH/GSSG
mutual transformation, synthesis of phytochelating peptides (PC),
ASA-GSH cycle and metabolism and other physiological activities in
Microcystis aeruginosa have interactions, which play a key role in
maintaining the antioxidant properties of microalgae and regulating
redox-sensitive signal transduction.

\textbf{Keywords:} Metabolic mechanism; GSH; Phycocyanin ; SOD; CQD;

\textbf{Introduction}

Microcystis aeruginosa is the most common algae in water blooms, and can
release toxic and harmful algal toxins into the
environment\textsuperscript{{[}1-4{]}}. There are two kinds of
glutathione in algal cells, one is reduced glutathione (GSH), the other
is oxidized glutathione (GSSG).However, glutathione in algal cells
exists in the form of reduced glutathione (GSH) in most cases, and GSH
has high reducibility \textsuperscript{{[}5-8{]}}. Under certain
conditions, oxidized glutathione (GSSG) and reduced glutathione (GSH)
can be transformed into each other \textsuperscript{{[}9-11{]}}. When
stressed, there will be reactive oxygen species (ROS) in microalgae
cells, which can cause GSH to be oxidized to GSSG
\textsuperscript{{[}12-14{]}}.As the most abundant free mercaptan in
microalgae cells, GSH maintains the best intracellular oxidation
environment to play the normal function of cell proteins
\textsuperscript{{[}15{]}}. In general, the level of GSH in microalgae
cells will affect the redox status of the
cells\textsuperscript{{[}16{]}}.

GSH in microalgae cells can also be converted into phytochelating
peptides (PC) and ascorbic acid (ASA) with the participation of various
enzymes \textsuperscript{{[}17-20{]}}. Under the action of
phytochelating peptidases (PCs), GSH can be synthesized into PC to form
low-molecular-weight complexes, which are then further converted into
high-molecular-weight multimeric complexes and preserved in Microcystis
aeruginosa cells \textsuperscript{{[}21{]}}. GSH exerts its important
antioxidant stress role through the ascorbic acid-glutathione (ASA-GSH)
cycle \textsuperscript{{[}22-24{]}}.

Therefore, under the influence of environmental changes, glutathione and
enzymes in algal cells undergo some synergistic changes and interact
with each other \textsuperscript{{[}25, 26{]}}, but with the progress of
metabolism, it is difficult to accurately detect and analyze the changes
of each living substance by existing biochemical methods. In this study,
we investigated the changes of GSH in different growth stages of
Microcystis aeruginosa and its correlation with the synergistic changes
of phycocyanin and enzymes, and analyzed the changes of carboxylated
biomolecules in the growth and metabolism of Microcystis aeruginosa by
covalent coupling method, mainly including the changes of GSH,
phycocyanin and SOD enzymes. The distribution of GSH content in
different stages was clear, no other interference in the system, good
correlation, changes in the number of peaks and peak area of GSH content
in the three-dimensional fluorescence spectral region, and the maximum
GSH content in the algal cells at d 50. There are physiological
activities between GSH synthesis, GSH/GSSG interconversion, synthesis of
phytochelating peptide (PC), ASA-GSH cycle and metabolism in algae. The
interaction between GSH synthesis, GSH/GSSG interconversion,
phytochelating peptide (PC) synthesis, ASA-GSH cycling and metabolism
was found to maintain the antioxidant properties of microalgae.

\textbf{2. Materials and Method}

\textbf{2.1Preparation and influencing factors of carbon quantum
dot-biomolecular conjugate}

(1) Preparation of conjugates \textsuperscript{{[}27{]}}: 1 mL of
water-soluble CQD solution was taken, EDC and NHS were added
sequentially to activate quantum dots, and finally biomolecules were
added to form a stable covalent conjugate. The resulting product was
stored at 4  for testing.

(2) Influencing factors \textsuperscript{{[}28, 29{]}}: The effects of
EDC amount, NHS amount, carbon quantum dot-biomolecular ratio, reaction
pH, and reaction time on the coupling reaction were investigated in a
certain range. The best reaction conditions were determined by comparing
and screening the fluorescence intensity.

a) Selection of coupling activator

Different concentrations of N-hydroxysuccinimide (NHS) were used as the
second activator. Stable intermediates are formed with carboxylated
quantum dots, which were then combined with biomolecules for coupling.
The optimal concentration was determined by fluorescence intensity.

b) Influence of concentration ratio on coupler preparation

Based on the pre-experiment, the amount of EDC, the amount of NHS and
the pH were kept unchanged, and the ratio of carbon quantum dots to
biomolecules was adjusted. The optimal concentration ratio was
determined by the fluorescence intensity of the conjugate.

c) Effect of reaction time on coupler preparation

The quantum dots were activated at different times (0h, 0.5h, 1h, 1.5h,
2h, 2.5h, 3h) and the optimal activation time was determined by the
fluorescence intensity of the quantum dots.

d) Effect of reaction pH

The conjugates were prepared at pH=5, 6, 7, 8 and 9, and the optimal pH
value was determined by the fluorescence intensity of the conjugates.

\textbf{2.2 Extraction of Glutathione, Phycocyanin and SOD from
Microalgae Cells}

As shown in Figure 1, Microcystis aeruginosa FACHB-905 was cultured on
days 7, 14, 21, 28, 35, 50, 65, 80 and 95, respectively. The algal
sludge was obtained by centrifugation at 4  and 8000 rpm for 10 min.

(1).Extraction of glutathione from algae cells: The broken algal cells
were added with 5\% sulfosalicylic acid to deproteinize the algal cells
in suspension state. The samples were centrifuged for 15 min and soaked
with 10 mL PBS for 2 h. After centrifugation for 20 min, the supernatant
was GSH extract.

(2). Extraction of phycocyanin from algae cells: Phosphate buffer at 4 
was added to algal mud samples in which Microcystis aeruginosa cells
were infiltrated before 4 . Freeze and thaw three more times. The
disrupted algal cells were suspended in acetone containing 40 mL of TCA
and DTT and precipitated at -20\selectlanguage{ngerman}°C for 2 hours. The samples were
centrifuged to discard the supernatant, precipitated and added to the
acetone solution, and repeated 2-3 times to obtain the phycocyanin
extract.

(3). Extraction of SOD from algae cells: The broken algal cells were
crushed in PBS phosphate buffer to infiltrate the algal cells, and the
supernatant was centrifuged to obtain the enzyme extract. The above
samples were extracted with 5 mL of chloroform-ethanol mixture to remove
various proteins. Finally, the precipitate was centrifuged and discarded
to obtain SOD enzyme liquid.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1 Extraction and analysis flow of microalgae GSH, phycocyanin and
SOD

\textbf{3. Results and discussion}

The absorbance at wavelength of 378 nm (OD378) of algal cells at
different growth stages at 7, 14, 21, 28, 35, 50, 65, 80 and 95 days and
the algal cell dry weight, observation of the microalgal growth process
by extending the culture cycle: Algal cells are in a phase of growth
adaptation from 0 to 14 days and grow more slowly; After entering the
second phase of logarithmic growth, the growth of algal cells was more
rapid, and both the absorbance OD378 and the dry weight of algal cells
showed a steady upward trend, the algal cells entered the third phase of
decay at 65 to 95 days, and the OD378 and the algal cell dry weight
gradually declined to the demise of the algal cells.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2 Growth and dry weight change of Microcystis aeruginosa

\textbf{3.1 Intracellular GSH changes}

The one-dimensional fluorescence spectra of GSH (Figure 3) showed that
the emission wavelengths of GSH detected by CQDs as probes were all
around 465 nm; from 0 to 14 d, the measured GSH content changed slowly
and increased significantly after 14 d, reaching the maximum on day 50.
The two-dimensional correlation of GSH content was calculated, and the
synchronous and asynchronous two-dimensional correlation fluorescence
spectra of GSH were obtained. It can be seen that there is only one
autocorrelation peak on the main diagonal, and there is no cross peak
outside the main diagonal. The excitation wavelengths of CQDs as probes
are all 370 nm, and the synchronous fluorescence change graph of GSH is
completely synchronous, i.e. all peaks of the same substance belong to
synchronous change. Since there is no obvious interference in the
system, no cross peaks exist outside the main diagonal. In the
two-dimensional asynchronous correlation fluorescence spectrogram, an
asynchronous cross peak appeared at 465 nm outside the main diagonal,
and excitation at 370 nm would get an emission peak at 465 nm, and the
distribution of GSH content at different stages was clear.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

Figure 3 Changes of GSH in algae cells at different growth stages (a)
and (b). Two-dimensional correlation fluorescence spectra (c)
.Two-dimensional asynchronous correlation fluorescence spectra and (d)

As Figure 4,the three-dimensional fluorescence spectrum of GSH shows
that during the growth period of microalgae, GSH is mostly in the
initial synthesis stage, so only one regional peak (region A) appears in
the contour plot and the peak area is small, and the peak height of GSH
in the three-dimensional fluorescence stereogram is low; On day 21,
Microcystis aeruginosa starts to enter the logarithmic growth period,
and the GSH in the algal cell starts to synthesize and transport, and
only one regional peak (region A) appears in the contour plot. On day
50, the intracellular GSH content reached its maximum, and the peak area
was the largest at this time, with three regional peaks, the outermost
region A, the middle region B and the innermost region C. The
three-dimensional fluorescence map showed the highest peak height of GSH
at this time. GSH is being synthesized, GSH/GSSG interconversion,
ASA-GSH cycle and synthesis of phytochelating peptide (PC) to play its
antioxidant role; Figure d enters the decay phase, the intracellular GSH
begins to metabolize and degrade, only two regional peaks (region A and
region C) remain and begin to decrease, and the peak height of GSH shown
in the three-dimensional fluorescence stereogram begins to decrease
Until the algal cells were completely dead, GSH was degraded to
L-\selectlanguage{greek}γ-\selectlanguage{english}glutamylcysteine (L-\selectlanguage{greek}γ-\selectlanguage{english}Glu-L-Cys, \selectlanguage{greek}γ-\selectlanguage{english}EC), \selectlanguage{greek}γ-\selectlanguage{english}glutamyl cycle potential
substrate, \selectlanguage{greek}γ-\selectlanguage{english}glycine cysteine and other free amino acids by the
catalytic action of carboxypeptidase (CPase) and \selectlanguage{greek}γ-\selectlanguage{english}glutamyl
transpeptidase (\selectlanguage{greek}γ-\selectlanguage{english}GTase). The distribution of GSH content at different
stages was clear, and there were no other interferences in the system.

\textbf{Hosted file}

\verb`image14.emf`  available at \url{https://authorea.com/users/623796/articles/646370-synergism-variation-between-intracellular-glutathione-phycocyanin-and-sod-in-microalgae-by-carbon-quantum-dot-fluorescence}

Figure 4 Three-dimensional fluorescence spectra and contour map of GSH
in algae cells on day 7 (a,b); on day 21 (c,d); on day 50(e,f); on
day(g,h)

\textbf{3.2 Changes in phycocyanin}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}

The phycocyanin content increased throughout the growth cycle, reaching
a maximum on day 65, and then began to decrease. Two-dimensional
correlation was calculated for the changes of phycocyanin content during
the growth cycle, and the synchronous and asynchronous two-dimensional
correlation fluorescence spectra of microalgae phycocyanin were
obtained. Figure 4.6(c) shows that there is only one autocorrelation
peak on the main diagonal, and there is no cross peak outside the main
diagonal. CQDs were used as probes to analyze the fluorescence changes
of phycocyanin content at different growth stages, and the excitation
wavelengths were all 370 nm, so no cross peak existed outside the main
diagonal because there was no obvious interference in the system. In
Figure 4.6(d), an asynchronous cross-peak appeared at 465 nm outside the
main diagonal. The asynchronous cross-peak appeared here reflected that
the excitation at 370 nm would get an emission peak at 465 nm, which was
consistent with the results shown in the previous 1D fluorescence
spectra, and the distribution of the algal cyanobacterial protein
content at different stages measured by the system was clear and well
correlated.

Figure 5 Variation of phycocyanin content in algal cells (a) (b); 2D
synchronous correlated fluorescence spectra (c); 2D asynchronous
correlated fluorescence spectra (d)

\textbf{3.3 SOD content changes}

As shown in Figure, SOD enzyme content showed an upward trend and
reached a maximum at 65 days. 2-D synchronous correlation fluorescence
spectra plots, it can be seen that there is only one autocorrelation
peak on the main diagonal, no cross peaks exist on the outside of the
main diagonal, the situation of the fluorescence change of SOD enzyme
content is probed by using CQDS as the probe, the excitation wavelength
is all 370 nm, the synchronous fluorescence change plot of SOD enzyme
all the peaks are synchronous changes; Since there was no significant
interference within the system, no cross peaks outside the main diagonal
existed. The asynchronous cross peak appears at 465 nm outside the main
diagonal, and the asynchronous cross peak presented here reflects that
excitation at 370 nm gives an emission peak at 465 nm, which is
consistent with the results shown in the previous one-dimensional
fluorescence spectrogram, and this system measures the SOD content
change at different growth stages, and the SOD enzyme content
distribution at different stages is clear and well correlated.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image23/image23}

\end{center}
\end{figure}

Figure 6 Changes in SOD enzyme (a) (b); Two-dimensional synchronous
correlation fluorescence spectra (c);Two-dimensional asynchronous
correlation fluorescence spectra (d).

\textbf{3.4 GSH metabolism and its correlation with phycocyanin, SOD}

GSH is an important substance involved in the regulation of algal cell
growth and metabolism, and as an important antioxidant in the process of
algal cell growth and metabolism, it participates in the metabolism of
the growth cycle in algal cells, and activates a variety of enzymes,
thus promoting the metabolic activities of proteins and enzymes, etc.,
in algal cells. By analyzing the correlation between two substances, GSH
and phycocyanin, the relationship between the methods of GSH and
phycocyanin detection was obtained based on the relative signal values
at different growth times: Y=0.4978X+0.0419, X indicates the relative
signal value of GSH and Y indicates the relative signal value of
phycocyanin, the maximum value of error is about 1.47 \% and the minimum
value is 0.46 \%,. For the correlation between GSH and phycocyanin in
algal cells during the growth phase, the correlation coefficient was
0.9925, which indicates a better correlation between GSH and
phycocyanin, when the fluorescence change values of GSH and phycocyanin
contents during the growth phase were then calculated.

By analyzing the correlation between the two substances of GSH and
SODase, and comparing the relative signal values, the relationship
between the methods of GSH and SODase detection was obtained as
Y=0.2869X+0.0702, X indicates the relative signal value of GSH and Y
indicates the relative signal value of SODase. The maximum value of
error was about 1.78 \%, while the minimum value was 0.09 \%, and the
correlation coefficient r was 0.9615, which means that the correlation
between GSH and SOD enzyme in algal cells was good.GSH is a tripeptide
with sulfhydryl group, which has an important role in reducing the
external oxidative damage to microalgal cells. GSH plays a key role in
the maintenance of antioxidant properties and the regulation of
redox-sensitive signaling in microalgae, and the level of glutathione is
associated with the resistance of microalgae to external stress. The
level of GSH is closely related to the tolerance of microalgae cells to
external stresses. The correlation suggests that as algal cells grow and
increase in vital material, intracellular GSH subsequently increases to
resist external oxidative slopes and environmental stresses.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image31/image31}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image32/image32}

\end{center}
\end{figure}

\textbf{Figure 7 The correlation between GSH and phycocyanin; GSH and
SOD enzyme}

GSH, together with SOD, acts as the core antioxidant factor of the redox
system, scavenging free radicals and preventing damage from peroxidation
reactions. Enhancement of GSH regulation by biological
\selectlanguage{greek}γ-\selectlanguage{english}glutamylcysteine synthetase promotes GSH regeneration.
\selectlanguage{greek}γ-\selectlanguage{english}glutamylcysteine synthetase (\selectlanguage{greek}γ- \selectlanguage{english}ECS) and glutathione synthetase (GS),
play key roles in the synthesis of GSH in Microcystis aeruginosa. \selectlanguage{greek}γ- \selectlanguage{english}ECS
can control the rate of GSH synthesis, and in turn GSH can also provide
feedback inhibition of \selectlanguage{greek}γ- \selectlanguage{english}ECS, as Fig.8.

(I)

(II)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image35/image35}

\end{center}
\end{figure}

Figure 8 Mechanism of GSH metabolism with enzyme in algal cells.

\textbf{4. Conclutions}

Using CQDs as fluorescent probes, we analyzed the changes of
carboxylated biomolecules in the growth and metabolism of Microcystis
aeruginosa by covalent coupling method, mainly including the changes of
GSH, phycocyanin and SOD enzymes in microalgae. The changes of GSH
content in Microcystis aeruginosa at different growth stages and the
correlation between phycocyanin, SOD enzymes and GSH in algal cells were
analyzed from one-dimensional, two-dimensional and three-dimensional
fluorescence, respectively. The correlation was good, and the number of
peaks and peak area of GSH content in the three-dimensional fluorescence
spectral region were changed, and the GSH content in the algal cells
reached the maximum at the 50th day. The GSH content of both phycocyanin
and SOD enzyme reached the maximum at day 65, and there was no other
interference in the system; GSH plays an important role in reducing the
external oxidative damage of microalgae cells. The interaction between
GSH synthesis, GSH/GSSG interconversion, synthesis of phytochelating
peptide (PC), ASA-GSH cycle and metabolism in Microcystis aeruginosa,
and the simultaneous linear changes between GSH and phycocyanin, SOD
enzyme changes all the time, played a key role in maintaining the
antioxidant properties of microalgae during the growth of microalgae and
in regulating redox-sensitive signaling. It plays a key role in the
regulation of redox-sensitive signaling during microalgal growth.

\textbf{Acknowledge}

This work was supported by Natural Science Foundation of Shanghai
(22ZR1460900)\textbf{.}

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