The synchronized motions of inner core residues allosterically modulate the activity of the protein kinase A catalytic subunit.

While heme synthesis requires the formation of a potentially lethal intermediate, protoporphyrin IX (PPIX), surprisingly little is known about the mechanism of its toxicity, aside from its phototoxicity. The cellular protein interactions of PPIX might provide insight into modulators of PPIX-induced cell death. Here we report the development of PPB, a biotin-conjugated, PPIX-probe that captures proteins capable of interacting with PPIX. Quantitative proteomics in a diverse panel of mammalian cell lines reveal a high degree of concordance for PPB-interacting proteins identified for each cell line. Most differences are quantitative, despite marked differences in PPIX formation and sensitivity. Pathway and quantitative difference analysis indicate that iron and heme metabolism proteins are prominent among PPB-bound proteins in fibroblasts, which undergo PPIX-mediated death determined to occur through ferroptosis. PPB proteomic data (available at PRIDE ProteomeXchange # PXD042631) reveal that redox proteins from PRDX family of glutathione peroxidases interact with PPIX. Targeted gene knockdown of the mitochondrial PRDX3, but not PRDX1 or 2, enhance PPIX-induced death in fibroblasts, an effect blocked by the radical-trapping antioxidant, ferrostatin-1. Increased PPIX formation and death was also observed in a T-lymphoblastoid ferrochelatase-deficient leukemia cell line, suggesting that PPIX elevation might serve as a potential strategy for killing certain leukemias.

The Haber-Bosch process, which synthesizes ammonia (NH₃) from nitrogen (N₂) and hydrogen (H₂), consumes approximately 2% of the global energy supply. A sustainable alternative is the direct electrochemical conversion of N₂ to NH₃. The selectivity and activity of the electrocatalysts for this process are assessed by quantifying the NH₃ present in the electrolyte. Compared with other analytical methods, ¹H NMR offers a straightforward approach for detecting NH₃ (by analyzing NH₄ ⁺). ¹H NMR method can also definitely confirm that the detected ammonia originates from the electroreduction of N₂ by comparing results obtained from isotopically labeled ¹⁵N₂ and regular ¹⁴N₂ gases. This capability is unique to the ¹H NMR method, as no alternative approaches offer this level of specificity. However, this method suffers from low sensitivity when measuring NH₄ ⁺ of low concentration of such as at μM or lower. To address this issue, we developed a novel approach that improves sensitivity by ∼3-fold through the introduction of ¹⁴N decoupling during the ¹H NMR data acquisition. Recently [Kolen M.ACS Omega2021, 6, 5698-5704], demonstrated a ∼3.5-fold increase in sensitivity by using a 1 mM concentration of the paramagnetic relaxation agent Gd³⁺. By combining our ¹⁴N decoupling technique with the relaxation agent Gd³⁺, we achieved a synergistic enhancement in sensitivity, resulting in an overall ∼10.9-fold sensitivity increase for the ¹H NMR detection of ¹⁴NH₄ ⁺. This translates to a reduction in NMR detection time by a factor of ∼119 (10.9²). This significant advancement enables the fast detection of ammonia at μM concentration or lower. ¹H NMR of ¹⁵NH₄ ⁺ with ¹⁵N decoupling was also demonstrated.

Polypropylene (PP) accounts for approximately 28.0% of the global polyolefin market, valued at $243.4 billion in 2022. Known for its lightweight, chemical resistance, cost-effectiveness, high strength and melting point, PP is widely used in various applications. Its properties and applications are closely tied to its tacticity. One-dimensional (1D) conventional ¹³C NMR has been extensively utilized to analyze PP tacticity, but its low sensitivity and longer relaxation time remain drawbacks. Typically, analyzing a single PP sample requires around 9 h of NMR time. Using of a cryogenically cooled 10 mm NMR probe can significantly reduce this measurement time; however, its high cost makes it inaccessible for most NMR laboratories. While the refocused insensitive nuclei enhanced by polarization transfer (RINEPT) technique is well-known for enhancing NMR sensitivity, there are no published studies using 1D ¹³C RINEPT to quantify PP tacticity. Relaxation agents like chromium (III) acetylacetonate (Cr(acac)₃) have also been used to reduce relaxation times in polyolefin NMR analyses. Here we introduce a straightforward and easily implementable 1D ¹³C NMR method for rapid PP tacticity quantification. This method combines Cr(acac)₃, Bruker's existing RINEPT pulse sequence (ineptrd), and our recently published ¹H decoupling sequence (bi_waltz65_256 pl) to eliminate ¹H decoupling artifacts. It is worth noting that decoupling artifacts are always present. When the signal-to-noise ratio (SNR) is low, these artifacts are obscured by noise. For example, in some two-dimensional (2D) or three-dimensional (3D) NMR spectra, decoupling artifacts are barely visible because of the low SNR. However, when attempting to observe weak signals in 1D spectra, increasing the sample concentration or the number of scans enhances the SNR, revealing the decoupling artifacts. The decoupling artifacts appeared superimpose with some other weak signals, affecting the measurements of signal intensities. Therefore, improved ¹H-decoupling methods are crucial for such data acquisition. This synergy results in a 9.4- to 9.7-fold sensitivity enhancement, equating to an 88- to 94-fold reduction (9.4² ≈ 88, 9.7² ≈ 94) in NMR acquisition time compared to conventional 1D ¹³C NMR experiment with Cr(acac)₃. The time savings are even more substantial compared to experiments without Cr(acac)₃. The faster and quantitative approach is accessible to researchers with or without cryoprobes. Beyond PP, this method can be applied to tacticity measurements of other polyolefins, such as polybutene, polyhexene and polyoctene.