It is possible that if a group in a bound substrate is exposed to the solvent surrounding the protein complex while the rest of the substrate is buried inside the protein, the pH of the solvent could affect the frequency of a Raman mode in which this group is involved?
The protein has amphoteric properties and its folding depends on the pH of the medium, the structure of the water. It can be observed by changing the Raman spectrum of water. See how it changes with the formation of micelles of dodecyl sulfate in our work
Raman spectroscopy of micellization-induced liquid-liquid fluctuations in sodium dodecyl sulfate
Yes, solvent pH will affect partially exposed protein bound substrate, which may be monitored by Raman spectroscopy, especially if the exposed chemical moiety is ionisable, i.e. can exchange a proton. Raman could be used to measure the pKa of the substrate, free in solution and bound to protein. It may be that such partially exposed groups will have their pKa altered upon binding, compared to being free in solution, via push - pull effects on the substrate electrons from the protein on the buried part of the substrate.
Substrate carbonyl groups are also susceptible to structure changes upon protein binding, which can be monitored by Raman spectroscopy - see Paul R. Carey's publications.
Solvent pH will affect both the exposed protein amino acids and bound partially exposed substrates, though those that are within the folds of the protein can behave differently, e.g. there pKa can shift from that usually observed when free in water, e.g. Protein folded and/or hydrogen bonded protein tyrosine can show a shift in pKa from that observed in water. The change in tyrosine molecular structure and bonding upon de/protonation can be monitored by Raman spectroscopy, as many vibrational modes are altered with changes in frequencies and intensities. Tyrosine has relatively intense Raman bands, which can be further enhanced using UV excitation [1].
Protein substrates that contain amides, carboxylic acid or hydroxyl groups often have their Raman spectra changed upon de/protonation, The pKa of such chemical moieties can alter upon protein binding, e.g. substrate binding to p-Hydroxybenzoate Hydroxylase can be monitored by Raman spectroscopy [2].
Substrates with "loose" electronic structure may also have their Raman spectra changed upon protein binding: some dyes change color upon protein binding, which indicates a change in structure or the equilibria between tautomers.
I hope the above helps: do you have a specific protein substrate complex you are studying?
1. J. Clarkson & D.A. Smith, “UV Raman Evidence of a Tyrosine in Apo-Human Serum Transferrin with a Low pKa that is Elevated upon Binding of Sulfate”, FEBS Letts 503(1), 30-34, 2001.
2. J. Clarkson, B. Pulfey & P.R. Carey, “Probing the Chemistries of Flavin Ring Systems of p-Hydroxybenzoate Hydroxylase by Raman Difference Spectroscopy”, Biochemistry, 36, 12560-12566, 1997.
When PH decrease protein are protonated affection Ranman frequency. Protonation also affect protein charge (negative) in COO- residual amino-acid group, which make it to aggregates.
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