Lactate dehydrogenase (LDH) is an enzyme which can be found in most major tissues. Serum levels of LDH are elevated in a wide variety of pathologic conditions, most notably cardiac and hepatic (liver) disease. LDH catalyses the formation of lactate from pyruvate and at the same time oxidises NADH to \( \mathrm{NAD}^{+} \). This is a reversible reaction as shown below. The measurement of LDH in the serum can be used to diagnose whether tissue damage has occurred. Students were tasked with the development of an assay for LDH activity in human serum samples from a control patient (healthy) and a patient who had suffered cardiac damage. Two samples were provided, labelled \( \mathbf{C} \) and \( \mathbf{D} \) (Control and Diseased serum). The students decided to follow the loss of absorbance of NADH in an appropriate assay buffer containing pyruvate as the substrate. Q1 (a) By examining the absorbance spectra of both \( \mathrm{NAD}^{+} \)and NADH below, comment why this approach is suitable strategy. [3]
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First, let's understand what NAD+ and NADH are. NAD+ (Nicotinamide adenine dinucleotide) is a coenzyme that plays a crucial role in various cellular processes, including energy metabolism and redox reactions. NADH is the reduced form of NAD+, which means it has Show more…
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Lactate dehydrogenase, an enzyme involved in glycolysis, from several species are compared using a Southern blot technique in the figure on the left: (A) yeast; (B) snail; (C) mouse; (D) rat; and (E) human (after K. Webster, Journal of Experimental Biology 2003). A. Justify the claim that these data provide evidence that supports glycolysis as a conserved core property. There are three forms of lactate dehydrogenase whose roles vary within an organism: LDH-A, -B, and -C. The question arises as to the origins of each. The cladogram on the right (after S. Tsuji et al., Proc Natl Acad Sci 1994) displays a proposed relatedness of variations in LDHs over many species. B. Based on these data, describe the differences between the likely evolutionary sequences of LDH-A, -B, and -C in the African frog and in humans. C. Evaluate the claim that in both species (African frog and human), LDH-C is the most recently evolved form of the enzyme.
The half-reactions involved in the lactate dehydrogenase (LDH) reaction and their standard reduction potentials are $$\begin{array}{l}\text { Pyruvate }+2 \mathrm{H}^{-}+2 r^{-} \longrightarrow \text { luctate } \quad \varepsilon^{0}=-0.185 \mathrm{V} \\\mathrm{NAD}^{\prime}+2 \mathrm{H}^{2}+2 \mathrm{c}^{2} \longrightarrow \mathrm{NADH}+\mathrm{H}^{+} \quad 8^{-1}--0.315 \mathrm{V}\end{array}$$ Calculate $\Delta G$ as $p H$ 7.0 for the LDH-catalyzed reduction of pyruvate under the following conditions: (a) [lactate]/[pyruvate] $=1$ and $\left[\mathrm{NAD}^{+}\right] /[\mathrm{NADH}]=1$ (b) $|$ lactate $|$ pynuvate $= 160$ and $\left|\mathrm{NAD}^{*}\right| /(\mathrm{NAD} \mathrm{H})=160$ (c) $|$ lactare $|$ / pynuvate $|= 1000$ and $\left|\mathrm{NAD}^{*}\right| /|\mathrm{NADH}|=1000$ (d) Discuss the effect of the concentration ration in parts a-c on the direction of the reaction.
The half-reactions involved in the lactate dehydrogenase (LDH) reaction and their standard reduction potentials are \[\begin{array}{ll}\text { Pyruvate }+2 \mathrm{H}^{+}+2 e^{-} \longrightarrow \text { lactate } & \mathscr{E}^{\circ}=-0.185 \mathrm{V} \\ \mathrm{NAD}^{+}+2 \mathrm{H}^{+}+2 e^{-} \longrightarrow \mathrm{NADH}+\mathrm{H}^{+} & \mathscr{E}^{\circ \prime}=-0.315 \mathrm{V}\end{array}\]Calculate $\Delta G$ at $\mathrm{pH} 7.0$ for the LDH-catalyzed reduction of pyruvate under the following conditions: (c) [lactate]/[pyruvate] $=1000$ and $\left[\mathrm{NAD}^{+}\right] /[\mathrm{NADH}]=1000$ (d) Discuss the effect of the concentration ratios in Parts a-c on the direction of the reaction. (a) [lactate]/[pyruvate] $=1$ and $\left[\mathrm{NAD}^{+}\right] /[\mathrm{NADH}]=1$ (b) [lactate]/[pyruvate] $=160$ and $\left[\mathrm{NAD}^{+}\right] /[\mathrm{NADH}]=160$
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