Arginine Amino Acids: Properties, Function, Benefits, and Sources

What is arginine?

L-Arginine (L-Arg) is an essential amino acid that plays a role in cellular development, ammonia detoxification, and the creation of creatine. L-arginine serves as a substrate for nitric oxide synthases (endothelial, inducible, and neuronal), wherein nitric oxide is produced through the cleavage of the amino group from L-arginine, exhibiting neurotransmitter and vasodilatory characteristics. A study by El-Hattab's team demonstrated the beneficial impact of l-arginine on mitochondrial function alterations, assessing the pharmacological effects of l-arginine and citrulline in individuals with MELAS syndrome. The participants were aged 18 to 57 years, clinically diagnosed with MELAS syndrome, and possessed a mutation in the MT-TL1 gene (3243A > G); the comparison group consisted of healthy persons aged 20 to 46 years. The study's results indicated that the supplementation of arginine (300 mg/day) and, more significantly, citrulline (300 mg/day) enhanced the rate of de novo nitric oxide synthesis and stabilized cellular respiration, thereby markedly decreasing the frequency of stroke-like episodes that precipitated migraine attacks, muscle weakness, and improved exercise tolerance.

L-arginine/nitric oxide pathway. (Hanusch B., et al., 2022)

Arginine structure

L-Arginine (2-amino-5-guanidino-pentanoic acid), characterized by its guanidino group (-C(NH2)2): This is a distinctive side chain feature of arginine. It comprises a core carbon atom that is doubly linked to nitrogen atoms, creating a guanidine structure, which is very basic and contributes to the basic qualities of arginine. The overall crystal arrangement in L-arginine can be characterized by highly puckered sheets with an average plane aligned with the ab-plane; this pronounced puckering results from the sinusoidal configuration of the chains that extend parallel to the b-axis. All hydrogen bonding in the structure transpires within these sheets (i.e., within the chains along the b-axis and the ribbons along the a-axis), while the stacking of the sheets along the c-axis is solely governed by van der Waals interactions.

Crystal structure of L-arginine. (Courvoisier E., et al., 2012)

Arginine pKa

Numerous critical metabolic reactions rely on the arginine side chain. Proteins and protein complexes rely on the planar guanidinium group for structural support, which it provides through ionic and hydrogen bonding interactions. Systems like bacteriorhodopsin and cytochrome c oxidase rely on it as part of their proton (H+) transport cascades. In these configurations, the completely protonated form of arginine regulates the proton acceptor and donor groups' pKa values. Additionally, arginine's guanidinium moiety is a nucleophile or electrophile for a range of post-translational changes, and it has also been suggested that arginine functions as a generic acid/base in a small number of enzymatic processes. The pKa value, which is the equilibrium acid dissociation constant, and the charge state of the arginine side chain are critical to all of these functions. Even when embedded in a very hydrophobic environment, like the interior of a protein or the midst of a lipid bilayer, an arginine in a quantitatively populated neutral state has never been conclusively detected within a biological context.

Because of its extremely polar guanidino group structure, arginine forms strong connections with water and charged molecules, which are critical in enzyme active sites and cellular settings. At physiological pH, the guanidino group can be protonated, creating a positive charge. This is essential for arginine's function in enzymes, particularly those that produce nitric oxide (NO), and for its involvement in numerous protein-protein interactions. A study employed complementary potentiometric and NMR spectroscopic techniques to reexamine the pKa value of the arginine side chain. The measured pKa values of 13.8 ± 0.1 are over 2 log units more than the values typically attributed to this residue. This discovery elucidates why, in contrast to other ionizable amino acids, arginines mainly retain a charged state in proteins under physiological conditions.

Arginine solubility

Arginine is a highly soluble amino acid in water. Its solubility is primarily due to its polar and charged functional groups, which can interact favorably with water molecules.

The stability of proteins over time, their concentration for structural research, and their storage at high concentration are all impacted by their unfolding and aggregation in solution. For aqueous protein solutions to be more stable, additives (also called cosolvents, cosolutes, excipients, etc.) are usually added. l-Arginine has several applications in the pharmaceutical industry, such as an eluent for protein recovery from an affinity chromatography column, an additive to prevent protein aggregation, and an enhancer of protein refolding. It is worth noting that Golovanov's team has demonstrated that three poorly soluble proteins with unrelated sequences—human MAGOH, fragment 1-153 of murine REF2-1, and WW domains 3 and 4 from Drosophilia Su(dx) protein (ww34)—have their maximum achievable concentrations significantly increased when l-Arg and l-Glu are added simultaneously in equimolar ratios. Studies have demonstrated that proteins can have their solubility increased by a factor of 4 to 8 when 50 mM of each additive, l-Arg and l-Glu, are mixed in an equimolar ratio. At the same concentration, the solubility was improved by less than 1.5 times when l-Arg and l-Glu were both dissolved in single-component solutions. The scientists postulated that l-Arg and l-Glu's charged side chains could prevent protein aggregation by interacting with oppositely charged residues on the surface of the protein and covering the adjacently exposed hydrophobic residues.

Preferential interaction coefficient values versus concentration for arginine and glutamic acid ions. (Shukla D., et al., 2011)

What does arginine do?

The uptake of L-Arginine by system y+ can be competitively inhibited by various cationic amino acids and positively charged analogues, including specific NOS inhibitors such as NG-monomethyl-L-arginine (L-NMMA) and NG-iminoethyl-L-ornithine. The manipulation of system y+ expression or activity, because to its significant role in L-arginine transport, constitutes a potential target for the modulation of cellular arginine metabolism.

In the majority of cells, L-arginine needs are predominantly fulfilled through the uptake of extracellular L-arginine via specialized transporters, including systems y+, b+, bo,+, Bo,+, or y+L. System y+ is expected to be the primary transcellular transporter of L-arginine, whereas other systems contribute minimally. System y+ is a high-affinity, Na+-independent transporter for L-arginine, L-lysine, and L-ornithine, with its function facilitated by members of the cationic amino acid transport (CAT) family, which includes at least four isoforms: CAT-1, -2A, -2B, and -3. The functions of particular transporters can be modulated in response to specific stimuli, including bacterial endotoxin and inflammatory cytokines, thereby enhancing substrate availability for augmented NO production.

L-arginine can produce nitric oxide by a five-electron oxidation process catalyzed by the enzyme nitric oxide synthase (NOS), which has three isoforms: constitutive neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and constitutive endothelial NOS (eNOS or NOS III).

Overview of L-arginine uptake and metabolism. (Ricciardolo F L M., et al., 2005)

Lysine vs arginine

L-arginine and L-lysine are naturally occurring basic amino acids, and their uses in the field of food science have garnered significant interest. Arginine is a semi-essential amino acid that facilitates wound healing, exerts immunomodulatory effects, and ameliorates symptoms associated with liver failure and obesity. Lysine is recognized as the "first essential amino acid" and is crucial for human growth and development. Over the past ten years, it has been shown that these fundamental amino acids can improve the characteristics of protein gels. Lysine engages in fewer electrostatic interactions within proteins and in a singular direction, whereas arginine participates in a greater number of electrostatic interactions in proteins across three potential orientations. A primary reason arginine remains charged under physiological settings is its elevated intrinsic pKa value. Historical tests spanning nearly a century frequently reference a side chain pKa value of approximately 12 in textbooks and structure-based electrostatics computations. The established pKa values of 13.6 and 13.4 for the conjugate acids of guanidine and N-methyl guanidine, respectively, strongly indicate that the intrinsic pKa of the arginine side chain is considerably greater than 12. Consequently, even when entombed in the most arid and hydrophobic settings, it may not undergo considerable deprotonation at neutral pH or at pH levels above 10. Consequently, due to an energy cost of ΔGo = 2.303RT(ΔpKa), the pKa of arginine is unlikely to decrease to a value as low as 7, as the host proteins would undergo unfolding due to the resultant loss of net stability. In this regard, the characteristics of the lysine side chain are markedly distinct. The intrinsic pKa value of approximately 10.4 for a random coil polypeptide indicates that the pKa values of lysines in folded proteins may be altered to below 7. Consequently, it is energetically feasible for buried lysine side chains to be entirely deprotonated and neutral at physiological pH.

pKa Values Obtained from Potentiometric Titrations. (Fitch C A., et al., 2015)

Arginine amino acids at Creative Peptides

Arginine benefits

L-arginine is a chemical with tremendous importance in quality assurance and clinical settings. It is mainly located in the regions of proteins where action takes place. Phosphate anion binding is facilitated by its structure, which allows it to catalyze phosphorylation processes. A large number of proteins rely on arginine to keep their charges stable. Arginine was degraded into urea and ornithine by arginase during nitrogen metabolism. Arginine is involved in immune system maintenance, hormone secretion, and ammonia detoxification. There is some evidence that nitric oxide synthase's ability to convert arginine into nitric oxide may improve vasodilation, which in turn may aid in the treatment of a number of physiological conditions, including cardiovascular disease, peripheral vascular disease, erectile dysfunction, atherosclerosis, vascular headaches, and chest pain. Protein synthesis is induced by arginine, which also aids in enhancing sperm production and prevents tissue wasting in critically ill patients.

Arginine uses

(1) As a cancer biomarker

Because some auxotrophic cancers can't grow without arginine, this amino acid can be used as a biomarker for these tumors. Because they lack the enzyme arginosuccinate synthase (ASS), malignant melanoma and hepatocellular carcinoma (HCC) cannot produce their own arginine. Angiogenesis is enhanced in tumors when tumor cells produce more nitric oxide, which in turn increases the demand for arginine. Arginine levels are decreased in leukemia patients compared to healthy individuals. Various kinds of cancer have plasma arginine concentrations that are higher than the usual range of 90-150 µM. For example, breast and colon cancer have 80±3 µM, pancreatic cancer has 76 ± 5 µM, and esophageal cancer has 41.9 ± 13.4 µM. In addition to reperfusion injury, asthma, arthritis, and psoriasis, a number of other clinical disorders have been associated with a markedly decreased arginine level as a result of increased arginase activity. So, it's clear that low arginine levels indicate physiological problems, which supports its importance as a biomarker.

Representation of arginine utilization by tumor cells. (Verma N., et al., 2017)

(2) Maintain the function of isolated heart

Using the rat heart as a model of ischemia-reperfusion injury, Agullo's research group examined the effects of arginine (L-Arg). Adding 3 mmol/l of arginine to the perfusate of the rat hearts that were isolated during the experiment reduced myocardial cell death and enhanced the release of cGMP. Based on their findings, the authors believe that the enhanced availability of cGMP during reperfusion is the primary mediator of the positive effect of L-Arg supplementation. In a study involving rats with spontaneous hypertension (SHR), Fujita and colleagues investigated the impact of oral administration of L-Arg for three weeks. We checked systolic blood pressure weekly. Blood pressure remained unchanged, they discovered. Perfusion of Krebs-Henselite solution in a Langendorff preparation was performed on the removed heart following the therapy. Utilizing a drop counter, the perfusion flow was quantified. The solution was stabilized before adenosine was administered to maximize vasodilation of the coronary arteries. We measured the change in coronary perfusion flow as a percentage after administering 10−4 M L-NMMA. The ablation of the atria and aorta did not affect the weight of the myocardium, and cardiac hypertrophy remained unchanged. After 3 weeks of treatment with L-Arg, the basal coronary perfusion resistance (CPR) in SHR remained unchanged. In contrast to the control SHR group, the SHR treatment group had a more pronounced L-NMMA produced CPR response. The researchers found that coronary flow reserve was reduced in SHR, but it was recovered after administering L-Arg.

The multiple biological roles of L-arginine. (Cziráki A., et al., 2020)

(3) Regulate blood pressure, blood flow, and endothelial function

L-arginine supplementation has been shown to enhance endothelial function in both animal and human patients. Experimental investigations indicate that L-Arg supplementation may be advantageous post-coronary angioplasty, attributed to reduced vascular remodeling and neointimal thickening due to the perivascular administration of this amino acid. The impairment of the L-Arg-eNOS pathway contributes to the pathophysiology of coronary artery disease (CAD). L-Arg has been shown to contribute to both the prevention and progression of CAD. Numerous human and experimental research examine the impact of L-Arginine treatment on vascular health, considering that this amino acid serves as the substrate for nitric oxide synthase, thereby facilitating nitric oxide synthesis, which is crucial for sustaining optimal endothelial function.

In 1992, the hypotensive effects of intravenous L-Arg were demonstrated by Hishikawa's group in normotensive persons. The participants' mean arterial pressure decreased from 79.3 ± 3.9 to 68.8 ± 2.2 mmHg, but their heart rates increased by about 8%. The concentration of nitrite/nitrate in the urine increased by 142.1 ± 12.4% compared to the baseline value when L-Arg was administered, and the plasma concentration of cGMP also increased.

Long-term administration of L-Arg to hypercholesterolemic rabbits increased endothelial-dependent vasodilatation and decreased intimal thickness and extent of atherosclerotic lesion in the thoracic aorta, as initially demonstrated by Cooke's group. The hypercholesterolemic group had significantly lower ACh-induced maximal relaxation of the thoracic aortic ring compared to the control group and the group that received L-arginine-HCl in drinking water supplementation (61 ± 5% vs. 89 ± 2% vs. 73 ± 3% for the hypercholesterolemic group, 73 ± 3% for the control group, and p < 0.05 for the supplemented group). While the control animals did not show any lesion after intimal lesion staining, hypercholesterolemic rabbits had an area of around 40%. Hypercholesterolemic rabbits given L-Arg had an area of intimal lipid accumulation less than 10% of the total. Supplementing hypercholesterolemic rabbits with L-Arg led to an 86% reduction in intimal thickness, with the greatest effect in the distal region of the artery.

(4) Refolding of recombinant proteins with arginine

The reducing environment in E. coli cells prevents the formation of disulfide bonds, causing unfolded proteins to aggregate into IBs, when heterologous proteins with disulfide bonds are produced in the cytoplasm of the bacteria. Disulfide linkages in expressed proteins are frequently erroneous even when proteins are inadvertently oxidized in the cytoplasm. Refolding the proteins into their native disulfide-bonded structure is necessary in both cases. Nowadays, the in vitro refolding solvent typically contains 0.1 to 2.0 M arginine with a pH range of 8.0 to 8.5. Created for single-chain antibodies (scFv), other immunoglobulin-folded proteins, and a number of interleukins, the additives-introduced stepwise dialysis refolding (AISD) method has meticulously investigated the function of arginine. Stepwise dialysis allowed for full refolding as shown by spectroscopic measurements performed during the process, which involved regulated coupling of the actions of oxidized glutathione and arginine. It seems that partially folded polypeptides with free thiol groups can be kept soluble by adding arginine to the refolding solution, which also stops the formation of disulfide bonds.

The effects of arginine on protein refolding. (Tsumoto K., et al., 2004)

(5) Solubilization of insoluble particles with arginine

When produced in E. coli, the majority of disulfide-containing proteins give rise to rigid inclusion bodies (IBs), which are commonly referred to as "classical" IBs. Soluble proteins can be produced in the cytoplasm of E. coli by expressing proteins that do not need disulfide bonds for folding, such as intracellular proteins when expressed spontaneously. On the other hand, expressed proteins tend to disperse into both the soluble and insoluble fractions, or they may be present exclusively in the latter. This structurally distinct family of insoluble pellets is known as loose IBs or "floccule-type" IBs. Overexpression or toxicity of the produced proteins could be the cause of this aggregation. In order to get the most out of the soluble fraction, two methods are typically employed. One strategy is to use a fusion system, alter culture parameters like temperature and medium, or coexpress chaperone proteins in order to boost expression. The second strategy involves adjusting the lysis conditions to boost recovery. The amount of expressed protein in the soluble fraction is increased when 0.1−1 M arginine is added to the lysis buffer, but our observation is only qualitative (T.A., unpublished results). On the other hand, native proteins can be recovered by extracting the pellet fraction of cell lysates using a solution that contains arginine. Figure 3 displays the outcomes that were achieved using this method. Solubilization of green fluorescent protein (GFP) from insoluble particles (i.e., floccule-type IBs) was demonstrated in this example experiment using 0.5, 1, and 2 M arginine. Higher concentrations of arginine resulted in increased recovery of GFP. Upon arginine extraction, GFP became luminous, suggesting that pellets had native or nearly native structures for some of the GFP molecules. So, contrary to the idea that IBs are inactive aggregates or the result of partially folded structural intermediates, a native and active protein could be extracted from IBs by adding arginine. Extraction of GFP using 0.5−2.0 M guanidium hydrochloride (GdnHCl) also resulted in luminous GFP. This suggests that 2.0 M GdnHCl does not denature GFP, which agrees with the observation that 4 M GdnHCl is necessary for GFP unfolding. At low concentrations (e.g., 2 M) of GdnHCl, hyperthermophilic archaeon proteins that are extremely heat-resistant can be dissolved. Similarly, arginine can dissolve the IBs of a variety of proteins, including those from archaeons, immunoglobulin-folded proteins, and cytokines (K.T. and M.U., unpublished observations). Based on these findings, it seems that arginine can dissolve most intractable particles. While it's true that both GdnHCl and arginine can dissolve IBs, it's important to note that arginine does not denaturate proteins at 2 M and does not destabilize proteins, while GdnHCl is a denaturant and destabilizes proteins even at concentrations without denaturing them.

(6) Suppression of protein aggregation by using arginine

One very unstable protein is ciliary neurotrophic factor (CNTF). When kept at 37 °C, it easily clumps together. When incubated at 37 °C, arginine effectively decreases CNTF aggregation. Therapeutic protein formulations, such as tissue-type plasminogen activator, make use of arginine. Although there was little change in the melting point of lysozyme as a function of arginine content, the reversibility of thermal melting was enhanced. Arginine inhibits bovine serum albumin (BSA) aggregation because, as the quantity of arginine increased, the temperature at which the protein seemed to begin melting (as measured by the presence of turbidity) rose.

(7) Attenuation of chilling injury for fruit

The most significant problem with storing cucumbers at low temperatures is chilling injury, which has a huge impact on the fruit's aesthetic quality and commercial value. For 16 days, cucumber fruits were preserved with varying doses of L-arginine (0.0, 0.5, 1.0, and 1.5 mM). The fruits were taken out of storage every four days, then conditioned for one day at a temperature of 22 ± 2 °C before being analyzed for quality. Various parameters were assessed in cucumber fruits, including their appearance, color, decay, weight loss, hardness, chilling injury (CI), electrolyte leakage, flavor, aroma, texture, soluble solids content (SSC), titratable acidity (TA), sugar: acid ratio (SSC/TA ratio), and ascorbic acid content. Fruits treated with L-arginine retained more of their original flavor, texture, scent, sugar-to-acid ratio, and overall fruit weight while exhibiting less electrolyte leakage and other storage-related issues. Fruits preserved their color, firmness, and flavor after treatment with 0.5 mM L-arginine, and their ascorbic acid content was higher than that of the control group. They also showed a considerable decrease in chilling injury and decay. In conclusion, pre-storage administration of L-arginine (0.5 mM) is a potential strategy for reducing chilling harm after harvest and keeping cucumber fruit quality while stored in cold conditions.

The storage life of persimmons is somewhat limited because they are climacteric, or perishable fruits. More and more focus in postharvest research has been on employing natural substances that are safe for both humans and the environment. The purpose of this study was to determine whether persimmon fruit could be effectively treated with 0, 0.3, or 0.6 mM of L-arginine after harvest to increase its chilling tolerance and preserve its nutritional value while stored at low temperatures. The results showed that the control fruit had the greatest levels of malondialdehyde (MDA) (5.8 nmol/g FW) and hydrogen peroxide (H2O2) (22.33 nmol/g FW), as well as the maximum weight loss (4.3%). Fruit treated with L-arginine had a slower reduction in firmness after storage, but the overall trend was the same. Fruit treated with 0.6 mM L-arginine showed the maximum tissue stiffness, measuring 3.8 kg cm−2. During storage, the chilling was progressively turned up. Chilling injury was less severe in fruits treated with L-arginine compared to the control group. While kept in the fridge, persimmons lost some of their antioxidant enzyme activity and total soluble tannin compounds. Antioxidant enzyme activity, antioxidant capacity, and total soluble tannin compounds were all considerably preserved by L-arginine treatment, although titratable acidity and total soluble solids were unaffected. It appears that numerous defense mechanisms generated by exogenous L-arginine contributed to a decrease in oxidative damage and an improvement in persimmon quality during low-temperature storage. According to these results, L-arginine is a great tool to have on hand for cold storage of persimmons since it helps to preserve their quality and extends their shelf life.

References

1. Hanusch B., et al., Characterization of the L-arginine/nitric oxide pathway and oxidative stress in pediatric patients with atopic diseases, International Journal of Molecular Sciences, 2022, 23(4): 2136.
2. Courvoisier E., et al., The crystal structure of l-arginine, Chemical Communications, 2012, 48(22): 2761-2763.
3. Fitch C A., et al., Arginine: Its pKa value revisited, Protein science, 2015, 24(5): 752-761.
4. Evich M., et al., Effect of methylation on the side-chain pKa value of arginine, Protein science, 2016, 25(2): 479-486.
5. Ricciardolo F L M., et al., The therapeutic potential of drugs targeting the arginase pathway in asthma, Expert opinion on investigational drugs, 2005, 14(10): 1221-1231.
6. Shukla D., et al., Understanding the synergistic effect of arginine and glutamic acid mixtures on protein solubility, The Journal of Physical Chemistry B, 2011, 115(41): 11831-11839.
7. Verma N., et al., L-arginine biosensors: A comprehensive review, Biochemistry and biophysics reports, 2017, 12: 228-239.
8. Cziráki A., et al., L-arginine-nitric oxide-asymmetric dimethylarginine pathway and the coronary circulation: translation of basic science results to clinical practice, Frontiers in pharmacology, 2020, 11: 569914.
9. Tsumoto K., et al., Role of arginine in protein refolding, solubilization, and purification, Biotechnology progress, 2004, 20(5): 1301-1308.
10. Hasan M U., et al., Pre-storage application of L-arginine alleviates chilling injury and maintains postharvest quality of cucumber (Cucumis sativus), J Hortic Sci Technol, 2019, 2(4): 102-108.
11. Nasr F., et al., Attenuation of chilling injury and improving antioxidant capacity of persimmon fruit by arginine application, Foods, 2022, 11(16): 2419.