Methods of Measuring Enzyme Activity Ex Vivo and In Vivo

Abstract

Enzymes catalyze a variety of biochemical reactions in the body and, in conjunction with transporters and receptors, control virtually all physiological processes. There is great value in measuring enzyme activity ex vivo and in vivo. Spatial and temporal differences or changes in enzyme activity can be related to a variety of natural and pathological processes. Several analytical approaches have been developed to meet this need. They can be classified broadly as methods either based on artificial substrates, with the goal of creating images of diseased tissue, or based on natural substrates, with the goal of understanding natural processes. This review covers a selection of these methods, including optical, magnetic resonance, mass spectrometry, and physical sampling approaches, with a focus on creative chemistry and method development that make ex vivo and in vivo measurements of enzyme activity possible.

Keywords: MALDI mass spectrometry imaging; electroosmotic push-pull perfusion; electroosmotic sampling; fluorogenic; magnetic resonance; microdialysis.

Figure 1.

Protons differ in their ability to exchange excited protons with water, their relaxivity (20). Exciting high relaxivity protons on a compound (panel a) has the effect of indirectly exciting water protons because of proton exchange. The signal from water thus appears more saturated when the said compound is present and being excited in comparison to the absence of the compound. A negative peak in the “%water signal” appears at the chemical shift (with respect to water) of the high relaxivity proton (panel c). When the high-relaxivity amide is converted to the lower relaxivity amine, the effect decreases (panel b). The phenolic proton has high relaxivity but at a different chemical shift and its relaxivity is relatively unaffected by hydrolysis of the amide (panel b). This proton acts as an internal standard or control against which the amide proton signal can be compared. Adapted from Sinharay et al. 2017 Magnetic Resonance in Medicine 77: 2005–2014. Copyright 2016 International Society for Magnetic Resonance in Medicine.

Figure 2.

Comparison of DDA steps in linear (a) versus spiral (b) MSI. In both cases, Step 1 is a full scan MS for the desired mass range and Steps 2–4 are sequential fragmentations of the 3 most intense ions in Step 1. The total ion chromatograms (TIC) of blue crab commissural ganglion tissue slices illustrate how spatial resolution is maintained with the spiral (d) method compared to the linear (c). Using the spiral method (f & h), the authors were able to map the distribution of the peptides HL/IGSL/IYRamide (m/z 844.4788) (e & f) and VSHNNFLRFamide (m/z 1132.6010) (g &h) whereas the linear method (e & g) revealed only a fraction of areas where the peptides were located. Reprinted with permission from OuYang, et al. 2015 Journal of the American Society for Mass Spectrometry 26: 1992–2001. Copyright 2015 American Society for Mass Spectrometry.

Figure 3.

Mass spectroscopic images of rat brain slices containing exogenously applied dynorphin B (DynB) in the absence (left) and presence (middle, right) of inhibitors. While Dyn B was hydrolyzed throughout the regions sampled, the fragments DynB (1-7) were more prevalent in the cortex (cx, indicated by arrows) whereas Dyn B (2-13) showed the highest intensities in the striatum (str). The inhibitor opiorphin (OPI) blocked nearly all formation of DynB (1-7) and DynB (2-13) whereas phosphoramidon (PA) prevented the formation of DynB (1-7) but did not appear to affect DynB (2-13) formation. The area of relatively higher intensity in the top, right corner of the DynB (1-7) images is due to a fold in the tissue. The bottom panel illustrates the tissue structures, including the corpus callosum (cc) used for region identification before (left) and after (right) matrix application. Reprinted with permission from Bivehed, E., et al. 2017. Peptides 87: 20–27. Copyright 2016 Bivehed, E. et al under Creative Commons license https://creativecommons.org/licenses/by-nc-nd/4.0/. Published by Elsevier Inc. DOI 10.1016/j.peptides.2016.11.006. Cannot be modified without permission from journal.

Figure 4.

Schematic showing a typical microdialysis experiment, in which the probe is implanted into the rat hippocampus. Inside the membrane, there is usually a concentric or side-by-side (shown) design of the inlet and outlet capillaries. Arrow indicates flow of perfusion fluid inside the membrane. Any species in the surrounding ECS, such as dynorphins and dynorphin fragments can diffuse through the membrane, be collected and quantified. C – cortex; CC – corpus callosum; HPC – hippocampus. Figure not drawn to scale.

Figure 5.

(a) Schematic of the online electroosmotic sampling experiment with microfluidic chip. The organotypic hippocampal slice culture (OHSC) sits upon a membrane insert on which it is grown. A sampling capillary is placed above the tissue while the other (distal) end is inserted into R5 reservoir of the microfluidic device. R1 contains ThoGlo-1, a thiol-specific reagent, R2 and R3 contains running buffer, and R4 contains Tris-HCl buffer. The thiol compounds collected from OHSC is allowed to react with ThioGlo-1 in the reaction channel, followed by separation via capillary electrophoresis. DP = detection point by laser-induced fluorescence. (b) Nonlinear fits of cysteamine (CSH) and pantetheine (P) generated from CoA hydrolysis. (c) Three proposed (I, II, III) degradation pathways of CoA. PSH = pantetheine. Adapted with permission from Wu et al. 2013. Analytical Chemistry 85: 3095–103 and Wu et al. 2013 Analytical Chemistry 85: 12020–27. Copyright 2013 American Chemical Society.

Figure 6.

Schematic illustrating a typical EOPPP experiment. (a) The CA1 and CA3 regions of the rat OHSCs are perfused with exogenous (b) Leu-enkephalin (YGGFL) and internal standard (^DY^DAG^DF^DL) at the same concentration, S₀, through a pulled fused silica capillary. YGGFL is hydrolyzed in the ECS by membrane-bound aminopeptidases with the major product being GGFL. YGGFL, ^DY^DAG^DF^DL and GGFL are collected in the sampling capillary and quantified offline using capillary liquid chromatography. (c) Numerical modeling gives estimates of the distribution of product concentration as a ratio to internal standard concentration which is equal to the initial substrate concentration, P/S₀. P/S₀ ratios under quasi-steady-state conditions are seen to vary considerably, making the assumption that the initial substrate concentration is constant untenable. (d) The integrated Michaelis Menten equation is fitted to P/S₀ produced at different S₀ to give estimates of V_max and K_m of aminopeptidase activity in the tissue. Adapted from Ou and Weber 2017. Analytical Chemistry 89: 5864–5873. Copyright 2017 American Chemical Society.