Consequence of macromolecular crowding on aggregation propensity and structural stability of haemoglobin under glycating conditions

We recorded the far- and near-UV circular dichroism (CD) spectra of solutions of α-chymotrypsin and sodium dodecyl sulfate (SDS) with the final surfactant concentration significantly above the critical micellization concentration. Solutions were prepared using three different procedures. The reference procedure was to mix the chymotrypsin solution with the SDS solution once, immediately achieving the final SDS concentration. In alternative procedures, the protein solutions initially contained some SDS and were mixed with pure SDS solutions at a concentration to provide the same final surfactant as the reference mixing. We demonstrate that the supplementation to the selected final concentration of SDS of the pure chymotrypsin solution leads to different CD spectra than the supplementation to this final concentration of SDS in the chymotrypsin solution containing a small concentration of a few millimolar SDS. These differences disappear when the initial concentration of SDS in the protein solution, which we then supplement to the indicated final concentration, is higher. This suggests the irreversibility of the processes caused by the addition of SDS to chymotrypsin and the influence of the initial amount of this surfactant on the processes occurring with its further addition to the solution. For quantitative analysis of far-UV CD spectra in terms of populations of protein secondary structure elements, we used four well-established software packages. All programs consistently indicate that the observed differences in the far-UV CD spectra can be explained by the differences in the increase in the population of helical forms in chymotrypsin under the influence of SDS.

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“…Moreover, the absence of absorption at 350 nm leads to the conclusion that there is no aggregation of CHA in our solutions. 34 , 35 …”

Section: Results and Discussionmentioning

confidence: 99%

Circular Dichroism Spectra of α-Chymotrypsin–SDS Solutions Depend on the Procedure of Their Preparation

et al. 2022

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“…Meanwhile, the bands between 450–700 nm are named as Q bands. The changes in the absorption of the Soret band are widely employed to evaluate the aggregation of alpha-1-microglobulin upon heme binding [ 63 ] and haemoglobin self-association in the presence of crowding agents and glycans [ 64 , 65 ].…”

Section: Uv-vis Absorption Spectroscopy and Turbidity Are Initial mentioning

confidence: 99%

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

2020

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The self-assembly of proteins is an essential process for a variety of cellular functions including cell respiration, mobility and division. On the other hand, protein or peptide misfolding and aggregation is related to the development of Parkinson’s disease and Alzheimer’s disease, among other aggregopathies. As a consequence, significant research efforts are directed towards the understanding of this process. In this review, we are focused on the use of UV-Visible Absorption Spectroscopy, Fluorescence Spectroscopy and Circular Dichroism to evaluate the self-organization of proteins and peptides in solution. These spectroscopic techniques are commonly available in most chemistry and biochemistry research laboratories, and together they are a powerful approach for initial as well as routine evaluation of protein and peptide self-assembly and aggregation under different environmental stimulus. Furthermore, these spectroscopic techniques are even suitable for studying complex systems like those in the food industry or pharmaceutical formulations, providing an overall idea of the folding, self-assembly, and aggregation processes, which is challenging to obtain with high-resolution methods. Here, we compiled and discussed selected examples, together with our results and those that helped us better to understand the process of protein and peptide aggregation. We put particular emphasis on the basic description of the methods as well as on the experimental considerations needed to obtain meaningful information, to help those who are just getting into this exciting area of research. Moreover, this review is particularly useful to those out of the field who would like to improve reproducibility in their cellular and biomedical experiments, especially while working with peptide and protein systems as an external stimulus. Our final aim is to show the power of these low-resolution techniques to improve our understanding of the self-assembly of peptides and proteins and translate this fundamental knowledge in biomedical research or food applications.

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“…63 It is appreciably accepted that in living systems, crowding vividly affects biochemical processes such as activity of enzymes and proteins' structural constancy. [64][65][66] Therefore, there is a need to understand this phenomenon of macromolecular crowding and its effect on protein aggregation and must be preferred to ape the "real life" situation in labs.…”

Section: Discussionmentioning

confidence: 99%

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding

Hasan

Naeem

2021

J of Molecular Recognition

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Macromolecular crowding plays an inevitable role in all biological processes influencing association, conformation, and other characteristics of proteins. Present study is based on the effect of macromolecular crowding on structure of horseradish peroxidase (HRP) enzyme. Concentration-dependent conformational changes induced by crowding agents, dextran 70 and polyethylene glycol (PEG)-4000, were monitored employing a range of biophysical techniques. The intrinsic fluorescence spectra showed transition of protein from native to unfolded state. Marked increase in 8-Anilino-1-naphthalene-sulphonoic acid and Thioflavin T fluorescence indicated presence of non-native moieties with 80 mg/mL dextran. Enhanced absorbance in turbidity, Soret, and Congo red in corroboration with scattering intensity at 350nm results revealed incidence of HRP aggregates. A new peak around 218 nm in CD spectra pointed towards change in secondary structure towards β-sheets. Significant loss of enzyme activity upon structural disruption was seen. Comet assay demonstrated DNA damage and genotoxic nature of HRP aggregates, supporting spectroscopic, and fluorescence results. The normalized resultswere obtained with 120 mg/mL PEG-4000 close to that of native HRP implying no disruptive effect on structure. It can be hypothesized that macromolecular crowding is a vital element, which can have diverse effects. In this study, dextran 70 was observed to have pro-aggregatory effect while enhanced stability of native enzyme was witnessed with PEG. Hence, it can be stated that PEG has potentially better crowder as it helps retain the native enzyme structure. Routine addition of crowding agents is recommended if biological molecules are to be studied under more physiologically appropriate environments.

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Consequence of macromolecular crowding on aggregation propensity and structural stability of haemoglobin under glycating conditions

Cited by 14 publications

References 67 publications

Circular Dichroism Spectra of α-Chymotrypsin–SDS Solutions Depend on the Procedure of Their Preparation

Circular Dichroism Spectra of α-Chymotrypsin–SDS Solutions Depend on the Procedure of Their Preparation

Evaluation of Peptide/Protein Self-Assembly and Aggregation by Spectroscopic Methods

The modulation of structural stability of horseradish peroxidase as a consequence of macromolecular crowding

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