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Influence of Macromolecular Crowding and Confinement On nalytic A al & B y i tr o Long, et al., Biochem Anal Biochem 2018, 7:2 s c i h e m m Biochemistry & Analytical e DOI: 10.4172/2161-1009.1000355 i h s c t r o i y B Biochemistry ISSN: 2161-1009 Research Article Open Access Influence of Macromolecular Crowding and Confinement on Enzyme Activity and Structure under Native and Denaturing Conditions Steven Long, Jeffrey Kunkel and Prashanth Asuri* Department of Bioengineering, Santa Clara University, Santa Clara, CA 95053, USA *Corresponding author: Prashanth Asuri, Department of Bioengineering, Santa Clara University, Santa Clara, CA 95053, USA, Tel: +14085513005; E-mail: [email protected] Received Date: Apr 04, 2018; Accepted Date: May 24, 2018; Published Date: Jun 01, 2018 Copyright: © 2018 Long S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract It is now well appreciated that both crowding and confinement influence enzyme structure and function due to excluded volume effects; however, the relative efficacies of these environments on protein fates remain unclear due to a lack of direct comparison studies. In this study, we explore the use of the biopolymer alginate to develop an in vitro platform to investigate the effects of both crowding and confinement on the behavior of two model enzymes - horseradish peroxidase and β-galactosidase. Alginate, in its solution phase, can be used as a crowding agent and, in its gel phase by crosslinking using divalent cations, to encapsulate and confine proteins, thereby allowing us to use the same system to directly compare the effects of crowding and confinement. Different degrees of crowding and confinement were achieved by varying the alginate concentration, and these studies demonstrated a clear dependence of enzyme activity on the degree of crowding and confinement. Moreover, our data also suggested that protein confinement in crosslinked alginate gels led to higher enhancements in enzyme activity under denaturing conditions relative to non-crosslinked crowded environments. Results from the kinetic analyses were corroborated using structural measurements of protein denaturation using the 8-anilinonaphthalene-1-sulfonic acid fluorescence assay. Keywords: Protein crowding; Protein confinement; Enzyme activity; protein fates, particularly using the same system and similar Enzyme structure; Protein denaturing conditions; Alginate solution measurement methods of protein structure and function [11,14]. It is and gel therefore of fundamental interest to develop experimental conditions to investigate and compare the effects of crowding and confinement on Abbreviations protein structure and function. HRP: Horseradish Peroxidase; β-gal: β-Galactosidase; ANS: 8- In this study, we report the use of the biopolymer alginate in order Anilinonaphthalene-1-Sulfonic Acid; ABTS: 2,2'-Azino-Bis(3- to establish conditions for experimentally distinguishing between the Ethylbenzothiazoline-6-Sulphonic Acid); ONPG: o-Nitrophenyl-β- effects of crowding and confinement, regarding their influence on Galactoside; GdnHCl: Guanidine Hydrochloride protein structure and function. Alginate was used since it is a high molecular weight, anionic polysaccharide that can act as a crowding agent in its solution phase; moreover alginate can also be crosslinked Introduction using divalent cations to encapsulate and confine proteins, thereby In vitro investigations of protein structure and function have been allowing us to use the same system to study the effects of both most commonly based on simple buffer systems containing low crowding and confinement [15-17]. Furthermore, as with other concentrations of protein that consequently do not sufficiently crowding agents, alginate concentration can be varied to control the replicate the highly crowded or confined intracellular environment of degree of crowding, as well as to control the pore size of the gel and proteins [1-4]. Recently, several studies have therefore investigated thereby the degree of confinement. The choice of alginate as a protein behavior using in vitro conditions that more closely simulate confining agent is relevant, as previous research has shown that the in vivo cellular environments, and supported that both although alginate gels exhibit large heterogeneity in pore size, most of confinement and crowding can significantly impact protein structure the pores range around 5-10 nm (especially for those prepared using and function [5-12]. While crowding and confinement are often used prepared using concentrations ≥1% w/v) [17-19]. In this study, we synonymously, there is a significant difference in how the two used three concentrations of alginate (1%, 5%, and 10% w/v) to study environments exert their influence on proteins. Crowding refers to the the roles of crowding and confinement on protein structure and presence of a high concentration of macromolecules that reduces the function. The influence on protein function under both native and volume of solvent available to the proteins, while confinement arises denaturing conditions was captured using kinetic assays on the model from encapsulation of proteins in spaces only moderately larger than proteins, horseradish peroxidase (HRP) and β-galactosidase (β-gal) – the proteins themselves [3,4]. Currently, theoretical analyses suggest two enzymes that have been routinely used to study the roles of various that the two environments differ in their extent to which they affect environments on protein function. These studies demonstrated a protein behavior [3,4,13]. Experimental studies support the theory; strong dependence of enzyme activity under native and denaturing however, the evidence has not been compelling, partly due to limited conditions on the degree of crowding and confinement. Similar results direct comparisons between the influences of these environments on were also observed for measurements of enzyme structure under Biochem Anal Biochem, an open access journal Volume 7 • Issue 2 • 1000355 ISSN: 2161-1009 Citation: Long S, Kunkel J, Asuri P (2018) Influence of Macromolecular Crowding and Confinement on Enzyme Activity and Structure under Native and Denaturing Conditions. Biochem Anal Biochem 7: 355. doi:10.4172/2161-1009.1000355 Page 2 of 7 denaturing environments using the 8-anilinonaphthalene-1-sulfonic ethanol was used based on our preliminary experiments that indicated acid (ANS) fluorescence assay (as further detailed in the Results that this concentration strongly inhibited enzyme function). We section). Furthermore, enzymes encapsulated in alginate gels were confirmed, by comparing the viscosity of alginate solutions prepared significantly more stable relative to enzymes in non-crosslinked using standard buffer and 35% ethanol, that the addition of ethanol did solution-phase alginate, when exposed to denaturing environments. not affect alginate solution properties. We also performed rheological Our data agrees well with previous theoretical investigations of the characterization of alginate gels soaked in buffer and 35% ethanol for effects of crowding and confinement on protein structure and ca. 30 min and did not notice any significant differences between the function, which suggest that confinement leads to greater elastic properties of buffer- and ethanol-soaked samples (data not enhancements in protein activity under denaturing conditions relative shown). For thermal denaturation experiments, the various enzyme to crowding [3,4,13]. To the best of our knowledge, this is the first formulations were maintained at 80 °C for different periods of time experimental investigation demonstrating a clear dependence of and then cooled to room temperature in an ice bath before measuring enzyme activity and structure on crowding and confinement using the the initial rates at room temperature. Enzyme assays were also same system. performed after incubating stock solutions of HRP and β-gal with 100 mM CaCl2 for 10 min to confirm that the addition of CaCl2 (for Materials and Methods enzymes in alginate gel preparations) did not impact enzyme reaction rates. We also confirmed, by exposing HRP- and β-gal-containing alginate gels to buffer solutions for different periods of time (up to 60 Materials min) that the enzymatic activities of the leachate was <10% of those of Horseradish peroxidase (HRP) and β-galactosidase (β-gal), as well the alginate gel-encapsulated enzymes (data not shown). as all reagents for the quantification of enzyme structure and function were obtained from Sigma Aldrich (St. Louis, MO). Alginic acid ANS fluorescence assays (sodium salt, low viscosity, 4-12 cP, 1% w/v in H2O at 25 °C) and calcium chloride for the crowding and confinement experiments were ANS fluorescence emission spectra (between 450 and 550 nm) of also purchased from Sigma Aldrich and used as received. enzymes in various alginate solution- and gel-phase conditions in standard Tris-HCl buffer conditions and denaturing conditions were collected after excitation at 360 nm using the Tecan Infinite 200 PRO Experimental setup for confinement and crowding studies spectrophotometer. The enzyme formulations were first equilibrated in For the confinement studies, enzymes were first encapsulated in either Tris-HCl buffer or chemical denaturant conditions (i.e. in buffer alginate before conducting the spectroscopic measurements. Briefly, solutions containing either 35% ethanol or
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