I am a novice in the field of fluoresence spectroscopy and I hope the pool of experts accumulated in research gate can help me. I have a transition metal complex bound to HSA and my tryptophan quenching experiment reveals that with increasing transition metal complex the fluoresence is more and more decreased. So far, so good! But other experiments revealed that the complex dissociates its ligands and only the transition metal is bound to HSA, which most likely binds (covalently) close to the Trp to a amino acid residue (and there maybe exhbiting some kind of distorted octahedral geometry). Since now I thought (because of my naivity and lack of knowledge) that the highest contribution to fluoresence quenching comes from the hydrophobicity of a bound ligand (or the kind of the bound ligand --> e.g. collision quenching) and/or an enviromental change of the binding pocket, where the Trp is located. However, the conformation is retained upon transition metal binding and no ligand is attached (--> no change in hydrophobicity within the pocket), so could someone please explain to me my quenching result? Is it sufficient to have only a single metal bound in the vicinity of Trp in order to observe quenching? The metal has the oxidation state +2/+3 and the initial complex ist octahedral.
Mr. Bijelic,
Toward your question, I personally have the following relating questions and few comments:
1. You have mentioned that a decreasing in intensity with an increasing in concentration of your complex is observed, but you have not mentioned have you change of band/s position/s or not?
You have only mentioned that there is not change of hydrophobic environment around Tyr and Trp chromophores, but if you have ligand exchange leading to coordination to HRS, as you have proposed, than you should have an effect of micro-environment. This effect should depend on the type of ligands in your initial complex.
The shifting effect is ca. 15 nm, by contrast to very broad both EAs and Fs of HSA and Trp, particularly. Trp has both Fs and Pl and the “shoulder” in the Fs is up to 410 nm. In this context:
2. Have you any chemometric data about the determination of Fs band positions and which are the values for errors and standard deviations?
Particularly in HSA there is overlapping between Fs of Tyr and Trp within 310-410 nm due to broad (and asymmetric) character of the bands. This yields to a significant increasing in error/s of measurements, including towards intensity and band position determination. In this context is the next question:
3. How much is this quenching effect?
4. Have you any temperature dependent measurements? If the quenching mechanism is a static one, than there is expected a decreasing in KSV with increasing in T. If it has dynamic nature, than KSV increases with increasing in T.
5. Why you have monitored your Fs experiments towards Trp, not and towards Tyr? Because of according to crystallographic data available [Ref. 1] (attachment), the active loop of HSA contains Tyrs residues (161Tyr and 138Tyr). This biomacromolecule is generally poor towards Trp (attachment). A parallel monitoring is obligatory aiming an accurate analysis of your system response.
6. Depending on type of your ligand within the frame of a ligand exchange process, you can have localization of those free initial ligands in different domains. Because of HRA shows different polarity at the different domains. This indirect effect could be associated with Trp-containing domain, where an eventual change of hydrophobic environment (but decreasing in this case) could lead to stacking effect with participation of 214Trp, which should cause for a decreasing in intensity in a static quenching mechanism. But this means that your ligands are aromatic, because of there is not aromatic amino acid residues around 241Trp one (Please pay attention to 1o9x.pdb (Ref.1)).
7. There is possible only interaction with a non-ligand exchanged complex ([Ref.1]) where only due to a change of hydrophobic character you have observe a quenching due to reason pointed out in point 4. But this depends also from the type of the ligands in your initial complex. But this means that you have interaction in a different active loop, containing 241Trp not 161Tyr/138Tyr as reported in [Ref. 1]. Please bear in mind as well as, however, that Trp-containing domain is not so polar, because of crystallographic data [ref.1] have shown a bonding of fatty acids around Trp, as well. Despite the fact that this complex [Ref.1] is also low polar molecular object.
In the context of points 1-7 a precise (unambiguous) assignment of coordination mode/s, i.e. molecular structure and behavior of your complex in HSA biomacromolecular matrix can be carried out using MALDI-MS of labeled ligands in your initial complex. You shall received direct information about a ligand exchange process and how your metal ions are coordinated; or which is their coordination environment. It there bonded Trp, furthermore covalently as you have proposed, what happen with the free exchanged ligands in the biological matrix and s.o.
Or, if you can isolate a single crystal and conduct crystallographic determination, than you shall unambiguously receive info about the structure and bonding/binding mode/s of your complex.
[Ref. 1] P. Zunszain, J. Ghuman, T. Komatsu, E. Tsuchida, S. Curry, Crystal structural analysis of human serum albumin complexed with hemin and fatty acid, BMC Struct. Biol. 2003, 3, 6
Dear Mrs. Ivanova,
thank you for your very detailed response! In the following I will shortly comment on your points 1-7:
1) Measuring fluorescence from 300-450 nm I have a very slight bathochromic shift (maximum 10 nm) of the peak (ca. 330 nm) at HSA:drug ratios of 5:1, 2:1 and 1:1, however, when the complex reaches molar excess over HSA (ratio 1:2 and 1:5) the peak is splitted into two peaks, where the "more bathochromic" peak is shifted by 20 nm (at 1:5 the fluorescence is slightly negative).
2) In this regard I am referring to published paper investigating similar complexes with HSA, however, as a novice I have to admit that I was unaware of the fact that "Tyr contribution" is that significant (The authors ignored this overlapping).
3) To explain it in words (I am not a fan of providing values): A twofold excess of the complex over HSA (HSA:complex 1:2) leads to a 50 fold decrease of the fluorescence compared to the apo-HSA flourescence.
4) Unfortunately not, but we are going to this in future. I am currently fighting with the method by applying the SV-plot to distinguish between both forms of quenching.
5) As mentioned before 'I didn´t took the "flourescence power" of Tyr into account (especially because these Tyrs are located within domain IB, wheras Trp is located at IIA)
6) My ligands are indeed aromatic, but I am quite sure that there is no ligand (I explain this later)
7) I don´t think that this is true for my case ....
AND NOW THE BOMB: I have solved the structure of this HSA-complex structure and there is no indication of an aromatic ligand in the Trp harboring pocket, only the metal center is bound to a residue close to Trp. However, in the electron density, the density of Trp is merged with the density of the coordination sphere of the metal. This means there is some kind of interaction. However, I cannot explain the outcome of the Quenching experiment since I do not believe that there is something hydrophobic (within the coordination sphere of the metal). Thus, there is a change in the micro-environment of Trp, but I guess it is something like water or chloride. Thus, I don´t really know what is causing the decrease in fluorescene ... the coordination of the metal? The small ligand which is coordinated to the metal and interacting with the Trp (which should be something polar like a ion or water)? Maybe there is some "lost" ligand swimming arround, but I cannot find it in the structure!
Mr. Bijelic,
Than the Fs quenching can be effect of the viscosity, due to different ionic strengths
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