Serum Albumin
Round 1
Reviewer 1 Report
Dear authors,
The manuscript is interesting entry about HSA which has been described many times in various review articles.
The strong point of the entry is a description of other HSA properties as usual - it means predominantly HSA as delivery system for many ligands which is the field of many articles in recent.
The authors focused on enzymatic, antioxidant, inflammatory properties as well as diagnostic and therapeutic applications of HSA when it interacts with endothelial cells. I consider this approach to be required and useful.
Reviewer´s recommendations:
- Insertion of some schematic figures into manuscript for better imagination of the text, e.g. a) the space structure of HSA into introduction which is about the structure or b) schematic figure about a trap of HSA within EGL should be very interesting......
- In introduction, molecular weights of serum albumins are missing.
- In section about the ability of HSA to treat OPs poisoning (row 220-225) would be mentioned that HSA molecule itself binds organophospate compounds (e.g. pesticides) such as written in paper: K. Želonková, S. Havadej, V. Verebová, B. Holečková, J. Uličný, J. Staničová: Fungicide Tebuconazole Influences the Structure of Human Serum Albumin Molecule, Molecules 2019, 24, 3190; doi: 10.3390/molecules24173190.
Minor revisions:
row 145: it´s should be its?
row 188 and row 220: the abbrevations CRP and OP are not explained.
row 220: The ability of albumin...
Author Response
We thank Reviewer-1 for the valuable comments on the manuscript. We have substantially modified the manuscript to address the points raised. The changes are detailed below and highlighted in the revised and improved manuscript.
Point 1
Insertion of some schematic figures into manuscript for better imagination of the text, e.g. a) the space structure of HSA into introduction which is about the structure or b) schematic figure about a trap of HSA within EGL should be very interesting......
Response 1
We have added a figure with 2d- and 3d-structure of albumin, Line 48. Since we have just five days for minor revision, we are not able to draw a scheme of HSA interaction with EGL. Instead, we have added a reference to the article that containes an illustration of this process, Lines 232-234:
“A detailed schematic illustration of the space between the plasma and the EGL with albumin molecules moving through the cleft is shown in Figure 1b of the work of Kundra et al. [81]”
Point 2
In introduction, molecular weights of serum albumins are missing.
Response 2
The information has been added, Lines 30-32:
“Molecular weight of HSA based on amino acid composition is 66.439 kDa, BSA – 66.267 kDa, RSA – 65.871 kDa, however the values of molecular weight can vary because of post-translational modifications and genetic variants.”
Point 3
In section about the ability of HSA to treat OPs poisoning (row 220-225) would be mentioned that HSA molecule itself binds organophospate compounds (e.g. pesticides) such as written in paper: K. Želonková, S. Havadej, V. Verebová, B. Holečková, J. Uličný, J. Staničová: Fungicide Tebuconazole Influences the Structure of Human Serum Albumin Molecule, Molecules 2019, 24, 3190; doi: 10.3390/molecules24173190.
Response 3
In the initial version of the article, we mentioned that albumin is able to interact with organophosphate pesticides (Line 44 in the initial version of the article). However, we have added some additional references, including Želonková et al., Line 58 in the revised version.
Point 4
Minor revisions:
row 145: it´s should be its?
Has been corrected: “its” instead of “it’s”
row 188 and row 220: the abbrevations CRP and OP are not explained.
There was the explanation for OP abbreviation, line 58 in the initial version if the article (Line 72 in the revised version). We have replaced CRP abbreviation by the full name of the protein: C-reactive protein (Line 240 in the revised version).
row 220: The ability of albumin...
Has been corrected
Reviewer 2 Report
This very well written entry fo enyclopedia is interesting and detailed. It gives a clear view on the field of research on albumins. I would like to suggest only a couple of minor improvements:
- the structural basis of albumin binding of many small molecules has been studied in very deep detail on human albumin, and more recently also bsa structure has been resolved. A sentence on strutural studies could be added to account also for this field of research.
- albumin exhibits many other very interersting catalytic activities, demonstrated in the framework of research on enzyme mimics. For instance, its ability to catalyze a benchmark reaction as the Kemp elimination was demostrated with a big impact in the litterature, and following the same thread, also the aldolase activity was observed, as well as the ability to control ketone reduction.
Author Response
We thank Reviewer-2 for the valuable comments on the manuscript. We have substantially modified the manuscript to address the points raised. The changes are detailed below and highlighted in the revised and improved manuscript.
Point 1
The structural basis of albumin binding of many small molecules has been studied in very deep detail on human albumin, and more recently also bsa structure has been resolved. A sentence on strutural studies could be added to account also for this field of research.
Response 1
The information about secondary and tertiary structure of HSA, BSA and RSA has been added (including Figure 1), Lines 32-37, 48.
“The secondary structure of the protein contains about 67% helical structures next to 33% of turn and extended chain configurations without any β-sheets [2] (Figure 1). The three-dimensional structure of HSA was resolved rather late, only in the 1990s [3]. A similar structure of BSA was obtained in 2012 [4], but the three-dimensional structure of albumin of rats, the principal animals used in pharmacological and toxicological experiments, has not been obtained yet.”
Point 2
Albumin exhibits many other very interersting catalytic activities, demonstrated in the framework of research on enzyme mimics. For instance, its ability to catalyze a benchmark reaction as the Kemp elimination was demostrated with a big impact in the litterature, and following the same thread, also the aldolase activity was observed, as well as the ability to control ketone reduction.
Response 2
Discussion about albumin enzymatic activities including Kemp elimination has been added, Lines 78-113:
“Bovine and human serum albumins catalyse the aldol reaction of aromatic aldehydes and acetone, with saturation kinetics and moderate and opposite enantioselectivity; the reaction occurs at the binding site in domain IIa, and is inhibited by warfarin [28]. A 101-amino-acid polypeptide derived from the sequence of the IIA binding site of HSA was identified, containing eight cysteine residues to form disulfide bridges that stabilize the polypeptide structure [29]. This protein retains the IIA fragment's capacity to bind typical ligands such as warfarin and efavirenz and other albumin's functional properties such as aldolase activity and the ability to direct the stereochemical outcome of a diketone reduction. It was suggested that some simple reactions that were catalysed by the serum albumin with Michaelis-Menten kinetics involve nonspecific substrate binding and catalysis by local functional groups [30]. These different active sites can bind promiscuously an array of hydrophobic negatively charged ligands, with a lysine residue acting as a primitive active site allowing these promiscuous activities to take place [31]. A method for predicting catalytic and substrate promiscuity using a graph-based representation known as molecular signature was suggested and enolase activity was among the first promiscuous activities described [32]. However, the binding mechanism via hydrophobic interaction with the binding site can be entirely different in presence of protein denaturing agent like urea, with electrostatic interaction playing a major role [33].
Most, if not all, enzymes are capable of catalysing physiologically irrelevant secondary promiscuous reactions in addition to the reactions that they have evolved to catalyse, and the universe of promiscuous activities available in nature was found to be enormous [34,35]. Nevertheless, we suggest that the promiscuity of albumin is principally different from that of the specialised enzymes, in that it was evolved as a result of not acquiring but a loss of some specialised activities, such as esterase (hydrolase) activities with digestive functions.
The Kemp elimination is regarded to be a prototypical reaction used to study proton abstraction from carbon. The reaction takes place at the so-called Stern layer, the interface between a micellar head or protein surface and water, and a significant rate accelerations can be achieved regardless of the precise positioning of substrates [36,37]. It is interesting to note that the reaction rate is decreased in protic solvents such as water as compared with aprotic organic solvents, the electrostatic term of the hydrogen bonds being the main factor for the large inhibitory effect of water; the presence of an external electric field oriented in the direction of the charge transfer increases the reaction rate [38]. On the other hand, the mechanism of the Kemp elimination in protein molecules was strongly associated with the presence of a catalytic base (Trp, Tyr, Phe) and a hydrogen bond donor (Lys, Arg, Ser, Tyr, His, water molecule) [39]. To this end, we were the first who explained the albumin mediated hydrolysis of some substrates by existence of catalytic dyads (as compared with catalytic triads in cholinesterases) His-Tyr or Lys-Tyr, where the histidine or lysine residues function as the acidic residues and hydrogen bond donors, whereas Tyr residue is a catalytic base [9].”
Round 2
Reviewer 1 Report
All minor revisions have been done so I recommend to publish the manuscript in Encyclopedia.
Reviewer 2 Report
The entry has been revised also according to my suggestions, in my opinion it can be published without any further revision.
