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Carbamylation is the non-enzymatic reaction between isocyanic acid and macromolecules (mainly proteins) which results in carbamylation-derived products (CDPs) generation, wherein the macromolecules show altered structure and function. In this study, we examined the modifications caused in human serum albumin (HSA) upon interaction with potassium cyanate (KCNO). HSA was incubated with varying concentrations of KCNO for 6 h at 37 °C. The resultant product was characterized by biochemical and biophysical techniques. Among other changes, the carbamylated-HSA showed homocitrulline generation (LC-MS), increase in mass (DLS), and amyloidogenic aggregate formation (Congo red, SEM, TEM). The Gibb's free energy was calculated to be −2.91 to −3.95 kcal mol−1, suggesting that the binding was spontaneous and energetically favourable. The results indicate that in chronic kidney disease patients, elevated levels of isocyanic acid (formed from urea) may modify the albumin structure and lead to its conversion into amyloidogenic aggregates, thus accelerating kidney damage.

Background Ornithine transcarbamylase deficiency (OTCD) is pleomorphic congenital hyperammonemia, in which the prognosis of the patient is determined both by genotype and environmental factors. This study investigated the clinical and biochemical characteristics of OTCD patients with different prognosis. Method Of 35 OTCD patients, six males deceased at the first disease-onset, 17 males survived and had controllable ammonia levels after treatment, and 12 females survived through the first disease-onset but had intractable hyperammonemia and high mortality. Fasting blood samples from patients collected at three disease stages were used for the analysis of amino acid (AA) profile, acylcarnitine profile, and micronutrients. Differences in profiles between patients and healthy controls and within patient groups were studied. Results All OTCD patients had accumulation of glutamine, homocitrulline, lysine, glutamate, cystathionine, and pipecolic acid, as well as deficiency of citrulline, tryptophan, threonine, and carnitine. For male non-survivors, most other AAs and long-chain acylcarnitines were elevated at disease onset, of which the levels of creatine, N-acetylaspartic acid, and homoarginine were remarkably high. Male survivors and female patients had most other AAs at low to normal levels. Compared with male survivors, female patients had much lower protein-intolerance, as indicated by significantly lower levels of protein consumption indicators, including essential AAs, 1-methylhistidine, acylcarnitines et al., but high levels of ammonia. Female patients still had significantly higher levels of citrulline, homocitrulline, and citrulline/arginine compared to male survivors. Conclusion Unique profiles were observed in each group of OTCD patients, indicating specific physiological changes that happened to them.

Rainbow trout and common carp are commercially important aquaculture fish species. To the best of our knowledge, the global composition of fish seminal plasma (SP) metabolites has never been studied. The aim of this study was to identify metabolites in carp and rainbow trout SP and to compare their relative abundances through the use of liquid chromatography-mass spectrometry (LC-MS)-based metabolic fingerprinting. Different solvents and sample/solvent ratios were tested for metabolite extraction. The largest number of metabolic features and the highest signal reproducibility were achieved using a 1:1 sample/acetone ratio for extraction. Accurate masses were searched against metabolite databases, and the identities of 48 and 40 metabolites for rainbow trout and carp SP, respectively, were confirmed using LC-MS/MS or through the standards. Most of the identified metabolites have not been previously described in fish SP. Several metabolites (amino acids, acylcarnitines, vitamins) were found in the SP of both species. However, differences between carp and rainbow trout SP metabolic fingerprints were observed. Certain metabolites appeared to be species-specific, e.g., propionic acid for carp and adipic and picolinic acids, homocitrulline and niacin for rainbow trout. Functional analysis revealed the involvement of SP metabolites in free radical scavenging, lipid and energy metabolism, molecular transport, cell death and survival, and inflammatory response. The identification of a high number of carp and rainbow trout seminal plasma metabolites provides new opportunities to develop novel biomarkers for sperm quality, optimise artificial reproduction and develop efficient cryopreservation procedures for these important aquaculture species.

Reduced skeletal muscle density in older adults is associated with insulin resistance, decreased physical function, and an increased all-cause mortality risk. To elucidate mechanisms that may underlie the maintenance of skeletal muscle density, we conducted a secondary analysis of previously published muscle composition and serum metabolomic data in 73 older adults (average age, 78 y). Multivariable-adjusted linear regression was used to examine associations between 321 metabolites with muscle composition, defined as the ratio between normal density (NDM) with low density (LDM) thigh muscle cross sectional area (NDM/LDM). Sixty metabolites were significantly (p ≤ 0.05 and q < 0.30) associated with NDM/LDM. Decreased renal function and the immune response have been previously linked with reduced muscle density, but the mechanisms underlying these connections are less clear. Metabolites that were significantly associated with muscle composition were then tested for their association with circulating markers of renal function (blood urea nitrogen, creatinine, uric acid), and with the immune response (neutrophils/lymphocytes) and activation (kynurenine/tryptophan). 43 significant NDM/LDM metabolites (including urea) were co-associated with at least 1 marker of renal function; 23 of these metabolites have been previously identified as uremic solutes. The neutrophil/lymphocyte ratio was significantly associated with NDM/LDM (β ± SE: −0.3 ± 0.1, p = 0.01, q = 0.04). 35 significant NDM/LDM metabolites were co-associated with immune activation. Carbamylation (defined as homocitrulline/lysine) was identified as a pathway that may link renal function and immune activation with muscle composition, as 29 significant NDM/LDM metabolites were co-associated with homocitrulline/lysine, with at least 2 markers of renal function, and with kynurenine/tryptophan. When considering that elevated urea and uremic metabolites have been linked with an increased systemic microbial burden, that antimicrobial defense can be reduced in the presence of carbamylation, and that adipocytes can promote host defense, we propose the novel hypothesis that the age-related increase in adipogenesis within muscle may be a compensatory antimicrobial response to protect against an elevated microbial burden.

In 2011 a novel autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, was described in rheumatoid arthritis (RA) patients. Anti-CarP antibody positivity associates with a more severe disease course, is observed years before disease onset, and may predict the development of RA in arthralgia patients. Although many clinical observations have been carried out, information on the antigenic targets of anti-CarP antibodies is limited. Most studies on anti-CarP antibodies utilize an ELISA-based assay with carbamylated fetal calf serum (Ca-FCS) as antigen, a complex mixture of proteins. Therefore, we analysed the molecular identity of proteins within Ca-FCS that are recognized by anti-CarP antibodies. Ca-FCS was fractionated using ion exchange chromatography, selecting one of the fractions for further investigation. Using mass-spectrometry, carbamylated alpha-1-antitrypsin (Ca-A1AT) was identified as a potential antigenic target of anti-CarP antibodies in RA patients. A1AT contains several lysines on the protein surface that can readily be carbamylated. A large proportion of the RA patients harbour antibodies that bind human Ca-A1AT in ELISA, indicating that Ca-A1AT is indeed an autoantigen for anti-CarP antibodies. Next to the Ca-A1AT protein, several homocitrulline-containing peptides of A1AT were recognized by RA sera. Moreover, we identified a carbamylated peptide of A1AT in the synovial fluid of an RA patient using mass spectrometry. We conclude that Ca-A1AT is not only a target of anti-CarP antibodies but is also present in the synovial compartment, suggesting that Ca-A1AT recognized by anti-CarP antibodies in the joint may contribute to synovial inflammation in anti-CarP-positive RA.

Background Homocitrulline (HCit) is a carbamylation-derived product (CDP) that has been identified as a valuable biomarker of morbidity and mortality in patients with chronic kidney disease (CKD). The aim of this study was to determine whether initiation of hemodialysis therapy (HD) could induce variations of HCit concentrations in CKD patients. Methods Serum HCit concentrations were determined by LC-MS/MS in CKD patients (n = 108) just before (M0) and six months (M6) after the initiation of HD therapy. Results Mean HCit concentrations reached 1000 μmol/mol Lysine before initiation of HD therapy and decreased by 50% within 6 months after HD onset. HCit concentrations remained stable over time as assessed during a 24-months follow-up period. HCit was mostly found in its protein-bound form in HD patients. HCit concentrations obtained at M0 were positively correlated with urea (r = 0.58) and carbamylated hemoglobin (r = 0.41), and are likely to be promising predictive markers of mortality. However, no correlations were found between HCit concentrations and Kt/V values, suggesting that HCit is not a marker of HD efficiency. Conclusion HCit concentrations reflect the intensity of protein carbamylation and are stable over time during HD treatment, making HCit a reliable biomarker in the follow-up of CKD patients.

Background Several investigators have reported discrepancies between the bromocresol-purple (BCP), bromocresol-green (BCG) and immunonephelometric (INP) assays in dialysis patients. This study compared the abovementioned assays and investigated whether hemodialysis itself or carbamylation of albumin is the cause for this discrepancy. Methods Samples obtained from hemodialysis patients were analyzed by BCP, BCG and INP. Furthermore, albumin was carbamylated in vitro using isocyanate. Isocyanate converts lysine to homocitrulline. Results No differences were observed between samples of the pre- and post-hemodialysis groups for BCP. In the control group, BCG averaged 6 g/L higher than INP. BCP did not statistically deviate from INP. In the dialysis group BCG averaged 5 g/L higher when compared to INP, whereas BCP averaged 2 g/L lower. BCP was affected by carbamylation of albumin. BCG and INP measurements were affected to a much lesser extent. Homocitrulline content of hydrolysates was increased in both the carbamylated albumin as well as in the dialysis population. Conclusion In a hemodialysis population albumin concentrations are not consistently estimated by both BCG and BCP methods. Relative to INP measurements BCG overestimates the albumin concentration (4–10 g/L), whereas BCP leads to an underestimation (0–4 g/L). Carbamylation of albumin is the main attributor to the discrepancy found with BCP.

Wheat gluten/glycerol (WGG) films were extruded with aqueous ammonia/salicylic acid or urea to investigate the reactions contributing to their hexagonal close-packed protein structures and material properties. The addition of aqueous ammonia and salicylic acid increased the pH, which, in turn, increased the level of intermolecular disulfide and lanthionine cross-links in the WGG films. Increased protein cross-linking reactions resulted in higher material strength and tensile modulus. These cross-linking reactions and the resulting material properties were similar for WGG films with 7.5 and 10% aqueous ammonia. Added urea into WGG film partially degraded into cyanate and ammonium. Cyanate subsequently reacted with lysine and cysteine to -carbamyllysine and S-carbamylcysteine, respectively. Even though these reactions resulted in a more alkaline reaction environment, hereby favoring disulfide bond formation and decreasing protein extractability, they also prevented the involvement of cysteine and lysine in protein cross-linking. The alkylation of these reactive amino acids, together with the plasticizing effect of urea, led to lower material strength and modulus with increasing levels of urea. publisher: Elsevier articletitle: Crosslinks in wheat gluten films with hexagonal close-packed protein structures journaltitle: Industrial Crops and Products articlelink: http://dx.doi.org/10.1016/j.indcrop.2013.08.077 content_type: article copyright: Copyright © 2013 Published by Elsevier B.V. ispartof: Industrial Crops and Products vol:51 pages:229-235 status: published

Aims Cerebellar ataxia is commonly observed in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disorder biochemically characterized by ornithine (Orn), homocitrulline (Hcit) and ammonia accumulation. Since the pathophysiology of cerebellum damage in this disorder is still unknown, we investigated the effects of Hcit and Orn on important parameters of redox and energy homeostasis in cerebellum of young rats. Material and methods We determined thiobarbituric acid-reactive substance (TBA-RS) levels, carbonyl content, nitrate and nitrite production, hydrogen peroxide production, GSH concentrations, sulfhydryl content, as well as activities of respiratory chain complexes I–IV, creatine kinase, Na + ,K + -ATPase, aconitase and α-ketoglutarate dehydrogenase. Key findings Orn and Hcit significantly increased TBA-RS levels (lipid oxidation), that was totally prevented by melatonin and reduced glutathione (GSH). We also found that nitrate and nitrite production was not altered by any of the metabolites, in contrast to hydrogen peroxide production which was significantly enhanced by Hcit. Furthermore, GSH concentrations were significantly reduced by Orn and Hcit and sulfhydryl content by Orn, implying an impairment of antioxidant defenses. As regards energy metabolism, Orn and Hcit provoked a significant reduction of aconitase activity, without altering the other parameters. Furthermore, Orn-elicited reduction of aconitase activity was totally prevented by GSH, indicating that the critical groups of this enzyme were susceptible to oxidation caused by this amino acid. Significance Taken together, our data indicate that redox homeostasis is disturbed by the major metabolites accumulating in HHH syndrome and that this mechanism may be implicated in the ataxia and cerebellar abnormalities observed in this disorder.

Background The number of newborns and the number of disorders detected by large-scale screening programs has increased dramatically in the last decade. Newborn screening is a multi-step process requiring confirmatory testing to establish and refine a diagnosis. Whereas screening cutoffs are established to detect all cases of a specific disease, confirmatory testing is performed against a backdrop of many co-morbid conditions and interpretation of results is often not straightforward. The goal of the current study was to define the range of amino acid concentrations encountered in normal, premature, and acutely ill newborns as an aid to the interpretation of follow-up testing for suspicious newborn screens. Materials and methods Residual plasma samples from 354 neonates (age 1–10 days) were utilized. 206 samples were from neonates with uncomplicated birth histories and prompt hospital discharge. 98 specimens were derived from a population of children receiving intensive care with diagnoses that included sepsis, respiratory insufficiency, cardiac malfunction/malformation and gastrointestinal complications. 50 samples were from infants born after 32 but before 37 full weeks gestation that were discharged following uneventful hospital courses. 25 amino acids were quantitated by LC-MS/MS and reference intervals determined by non-parametric statistical methods. Distributions were compared using Kruskal–Wallis analyses for independent samples. Results The distributions of multiple amino acids in premature and critically ill infants were significantly different than those observed in healthy newborns. Differing distributions were found for phenylalanine, the branched chain amino acids, methionine, glutamine, glutamate, arginine, lysine, α-aminoadipic acid, and β-aminoisobutyric acid. In most cases median values were increased and distributions more positively skewed in premature or ill newborns compared to healthy newborns. In addition, we report neonatal homocitrulline reference intervals for these newborn populations determined by an LC-MS/MS technique that is not confounded by methionine interference. Conclusion These data provide a basis for improved detection of genetic metabolic disorders in newborns, particularly those born prematurely or with a variety of critical co-morbid conditions.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is an autosomal recessive disorder caused by mutations in ORNT1 gene that encodes a mitochondrial ornithine transporter. It has variable clinical presentations with episodic hyperammonemia, liver dysfunction, and chronic neurological manifestations. In this work, we report the findings of HHH syndrome in 3 Saudi siblings. The 4-year-old proband presented with recurrent Reye-like episodes, hypotonia, and multiple stroke-like lesions on brain MRI. Biochemical and molecular analysis confirmed that she had HHH syndrome. She significantly improved on protein restriction and sodium benzoate. Her two older siblings have milder phenotypes with protein intolerance and learning problems. In comparison to their sister, their homocitrulline and orotic acid were only mildly elevated even before treatment. The three patients were homozygous for a novel mutation in ORNT1 with a Gly220Arg change. In view of the CNS lesions, which initially were felt to be suggestive of MELAS, we sequenced the entire mtDNA genome and no potential pathogenic mutations were detected. Analysis of ORNT2 did not provide explanation of the clinical and biochemical variability. This work presents a yet unreported CNS involvement pattern, notably multiple supratentorial stroke-like lesions in association with HHH syndrome. Moreover, it illustrates considerable clinical/biochemical correlation, and describes a novel mutation. We suggest including HHH syndrome in the differential diagnosis of patients found to have stroke-like lesions on brain MRI.

Carbamoylation of amino acids and proteins in uremia. Cyanate spontaneously transformed from urea increases as renal function decreased. Acting as a potential toxin, the active form of cyanate, isocyanic acid, carbamoylates amino acids, proteins, and other molecules, changing their structure, charge, and function. The resulting in vivo carbamoylation can modify the molecular activity of enzymes, cofactors, hormones, low-density lipoproteins, antibodies, receptors, and transport proteins. Antibodies specific for e-amino-carbamoyl-lysine (homocitrulline) located carbamoylated proteins in situ in neutrophils, monocytes, and erythrocytes. Carbamoylated proteins were found in renal tissue from uremic patients but not in normal transplanted kidneys. The irreversible reaction with cyanate converts free amino acids (F-AAs) to carbamoyl-amino acids (C-AAs). The Carbamoylation Index (CI), C-AA/F-AA, quantifies the decrease of the F-AA pool for each essential amino acid. C-AAs contribute, in part, to malnutrition of uremia. C-AAs interfered with protein synthesis to lower 14 C hemoglobin synthesis in human reticulocytes and osteocalcin synthesis in rat osteosarcoma-derived tissue culture. Insulin-sensitive glucose uptake was decreased 33% in cultured rat adipocytes by α-amino-carbamoyl-asparagine. e-Amino carbamoylation occurs primarily in F-AA, while e-amino carbamoylation of lysine in protein occurs continuously during the protein life span. Protein catabolism releases e-amino-carbamoyl-lysine. Quantitation of α versus e carbamoylation may yield a more sensitive measurement of protein intake versus protein catabolism, and could be useful in decisions concerning the time to initiate dialysis or subsequent changes in dialysis prescription. Carbamoylated molecules can block, enhance, or be excluded from metabolic pathways, thereby influencing the fate of noncarbamoylated molecules. Although not an "all-or-none" phenomenon, urea-derived cyanate and its actions are contributing causes of toxicity in uremia.

Polyclonal antisera to epsilon-amino-carbamoyl-lysine (homocitrulline) on guinea pig carbamoylated low density lipoprotein were used as probes to identify homocitrulline. These antisera detect homocitrulline in the immunogen and on other carbamoylated blood proteins including those from different species. In vivo carbamoylation of intracellular hemoglobin obtained from uremic patients and carbamoyl-proteins, carbamoylated in vitro, located on erythrocyte and lymphocyte cell surface membranes were detected. Solid phase, direct and competitive ELISAs for homocitrulline were used to analyze carbamoylated blood proteins. In uremic patients carbamoylation of protein by endogenous urea-derived cyanate occurs on the epsilon-amino group of lysine residues. Identification of carbamoylated proteins from uremic patients using an immunochemical probe of site specific antibody provides a way to monitor post-translational modification of proteins due to uremia and may give further insight for understanding the pathophysiology of end stage renal disease.

Analyis of amino acids is complicated by treatment with ampicillin. High voltage electrophoresis, which is convenient for the qualitative assessment of metabolic diseases, yields smears of ampicillin that mask the bands of citrulline, homocitrulline, phenylallanine, cystine, and homocystine. The addition of penicillinase prior to high voltage electrophoresis eliminates ampicillin and other penicillins and reveals these key amino acids.

1. 1. Radioactive homocitrulline has been identified in the urine of a rat following injection of [U-14C]lysine. 2. 2. Ornithine transcarbamylase was found to have a Km for L-lysine which was 10 times greater than that found for L-ornithine. 3. 3. Arginase was able to catalyze the breakdown of L-homoarginine, having a Km 200 times greater than L-arginine. 4. 4. Homogenates of rat liver or kidney failed to produce [U-14C]lysine during incubation with L-homo[U-14C-lysine]citrulline, indicating that L-lysine cannot replace L-ornithine in the urea cycle of mammals.

The alpha-keto acid analogs of arginine and citrulline (and of their next higher homologs, homoarginine and homocitrulline) were prepared enzymatically and shown to exist in equilibrium with cyclic forms which predominate in solution and in the solid state. The cyclic forms of the alpha-keto acid analogs of arginine and homoarginine have the structures pyrrolidine-1-amidino-2-hydroxy-2-carboxylic acid and piperidine-1-amidino-2-hydroxy-2-carboxylic acid, respectively. The cyclic forms of the alpha-keto acid analogs of citrulline and homocitrulline have the structures pyrrolidine-1-carbamyl-2-hydroxy-2-carboxylic acid and piperidine-1-carbamyl-2-carboxylic acid, respectively. The latter compound can undergo further cyclization and dehydration to form additional products, whose structures (2H, 5H, 7H-imidazo[1,5-a]pyridine-1,3-dione and 2H, 5H, 7H, 9H-9-hydroxy-imidazo[1,5-a]pyridine-1,3-dione) were deduced. The alpha-keto acids investigated here, which may be formed reversibly by enzymatic transamination of the corresponding amino acids, may be formed in vivo especially in the presence of metabolic abnormalities associated with inborn enzymatic defects.

The binding of [ 14 C]warfarin to normal human serum albumin (HSA), carbamoylated albumin, and plasma of patients with renal failure was investigated. Warfarin binding to extensively carbamoylated albumin (exhibiting a molar ratio of homocitrulline: albumin of ca . 7) was markedly diminished. The decrease in binding was apparently due to a decrease in the primary binding affinity constant. The K a for warfarin binding to HSA was 1.6 × 10 5 M −1 compared to 0.88 × 10 5 M −1 for carbamoylated HSA. Binding to normal plasma was significantly greater than to plasma from uremic patients. Mean fractions bound (±S.E.M.) were 98.0 ± 0.2 and 94.1 ± 1.6 respectively (P 14 C]warfarin to carbamoylated albumin with a homocitrulline: albumin molar ratio of 0.5 was marginally diminished, suggesting that the carbamoylation of albumin that occurs during renal failure contributes only slightly to the defective plasma binding of warfarin in uremia.

A child with a severe form of citrullinemia has been studied over four years. The biochemical findings included increasedblood levels of citrulline, alanine, glutamine, homocitrulline, and carnosine, and increased urinary content of citrulline, glutamine, orotic acid, uracil, homocitrulline, and N-acetylcitrulline. Urea synthesis was compromised. The beneficial effect of dietary restriction of protein is illustrated by the rate of head growth and by the subsequent development of the child. Heterozygotes may be detected by assay of fasting plasma citrulline levels. Enzyme studies on cultured skin fibroblasts indicated a marked lack of argininosuccinate synthetase activity in the patient and approximately half-normal activity in three heterozygotes.

Processing foods may cause unintended changes in their constituent proteins. Bovine milk protein studies have assisted in understanding the formation of lysinoalanine, which has toxic properties, and some phosphoproteins. Protein lysyl residues may enter the Maillard reaction with degradation products of carbohydrates, but losses of lysine can occur in the absence of free carbohydrate. Other alanines have also been detected. The study of bovine milk casein complex, via its four components, has yielded additional understanding of lysinoalanine formation, and has indicated that it corresponded to the lysine loss, but not to the serine loss. The studies report the effect of gentle heat and alkali at neutral pH, but greater heat at the same pH has yielded lysinoalanine. Cyanate, formed from milk urea, may lead to homocitrulline formation.

The LD50 and LD99.9 levels for ammonium acetate injected intraperitoneally in rats starved for 24 hr. have been determined. Injection of 2 mmoles/kg. body weight of l -arginine. HCl at 60 min. prior to an LD99.9 dose of the ammonium salt resulted in complete protection of the animals. A comparable degree of protection with l -citrulline and l -ornithine. HCl was achieved at a level of 8 mmoles/kg. l -Arginine methyl ester. HCl, neutralized to pH 7.0, conferred nearly complete protection at a level of 4 mmoles/kg. of body weight. Compounds which when injected 1 hr. prior to an LD99.9 dose of ammonium acetate protected some but not all of the animals included the d -isomers of arginine. HCl, citrulline, ornithine. HCl, and arginine methyl ester. HCl, as well as α-keto-δ-guanidovaleric acid (the α-keto acid analog of arginine) and α-acetyl- l -ornithine. Compounds which, under the same conditions, were completely nonprotective, included acetyl- l - and d -arginine, α-acetyl- d -ornithine, δ-acetyl- l -ornithine, and the l -forms of lysine, homocitrulline, and homoarginine. Liver slices prepared from animals injected 60 min. earlier with various protective compounds showed, when incubated with ammonium chloride, an accelerated consumption of ammonia and formation of urea, whereas with nonprotective compounds under the same conditons either a less strongly marked acceleration or no acceleration at all was observed. It would appear that the effect of the previously injected protective substances consisted at least in part in a mobilization and acceleration of the classic Krebs-Henseleit urea synthesis mechanism in the liver.

The metabolism of lysine, homocitrulline, and homoarginine in the isolated, perfused liver was investigated. Following the addition of 4 mmoles of lysine to the perfusate, homocitrulline but not homoarginine appears in the perfusate and the liver. The liver/perfusate concentration ratio for homocitrulline is approximately 4, an indication that homocitrulline is not rapidly transported out of the cell. Homocitrulline probably is not metabolized by the liver since the concentration of added homocitrulline remains constant during 4 hours of perfusion. The addition of 1-(guanido-14C)-homoarginine to the perfusate results in the appearance of labeled urea in the liver and perfusate. Apparently the reaction is carried out by liver arginase, since it is demonstrated that homoarginine is hydrolyzed by bovine liver arginase to form urea. The reactions suggest an alternate, minor pathway of ureogenesis in the organism.

1. Potassium cyanate, a reagent that carbamylates proteins, reacts both with pepsinogen and pepsin. 2. When allowed to react with pepsinogen, nine of the 10 e-NH2 groups of the lysine residues are carbamylated and homocitrulline is formed. In addition, reaction occurs with 4 tyrosine residues but not with the α-NH2-terminal leucine. 3. Carbamylation of pepsinogen is accompanied by a decreased susceptibility to activation. The enzymically active product that can be obtained is, in its amino acid composition and NH2-terminal amino acid, indistinguishable from pepsin prepared from the untreated zymogen. 4. Carbamylation results in a change of the molecular conformation of the protein as reflected by a decrease of λc from 236 to 220 mµ, an increase of the levorotation, [α]366, from -212 to -297, and a change of [m'] from -4086 to -3802. 5. Pepsinogen in which 3 lysines are carbamylated retains its native conformation and full potential pepsin activity. On further reaction with potassium cyanate, the loss of potential pepsin activity parallels the percentage change of the specific optical rotation, [α]366. 6. When pepsin is carbamylated, the proteolytic activity of the enzyme toward hemoglobin decreases, whereas the hydrolysis of the synthetic substrate, N-acetyl-dl-phenylalanyldiiodotyrosine, increases 2.8-fold. These changes can be reversed by decarbamylation with hydroxylamine. 7. With the aid of differences in the absorbance and in the incorporation of K14CNO it was established that 6 tyrosine residues were carbamylated in pepsin. Although the NH2-terminal isoleucine had reacted, the 1 lysine residue of the protein was not carbamylated.

1. 1.|The reaction of κ-casein B and α s 1 - casein C with formaldehyde suggested that their free amino groups might be essential for their native functions. Other amino group-modifying reagents were then employed since the specificity of formaldehyde is doubtful. 2. 2.|The capacity of κ-casein B for stabilizing α s 1 - casein C in the presence of Ca2+ is abruptly abolished after five of its nine positively charged lysine residues per molecule (monomer mol. wt., 19 000) are converted to uncharged homocitrulline residues by carbamylation. A marked decrease in sedimentation coefficient at 1% protein concentration coincided with the loss of protective colloid function of carbamylated κ-casein B. These findings indicate that changes in conformation and/or aggregation of κ-casein B occur due to the increase in net negative charge that accompanied carbamylation of the fifth lysine residue per molecule. 3. 3.|In other experiments the lysine residues of κ- and α s - caseins were converted to homoarginine, e-N,N- dimethyllysine and e-N- isopropyllysine residues. Such modification retains the charges of the lysine residues while sterically hindering their e-amino groups. Since the native properties of the κ-casein B and α s 1 - casein C were conserved, it seems doubtful that a specific e-amino group is critically involved in the interaction of these proteins.

Guanidination of the nine e-amino groups or picolinimidylation of the α- and e-amino groups yielded active derivatives of DNase. Since modification of the enzyme with these large substituents, which have delocalized positive charges, should have inactivated the protein if amino groups were involved in the catalytic reaction, none of the primary amino groups of DNase can be essential for catalysis. When positive charges on the enzyme were removed by carbamylation of the α-amino group and about seven e-amino groups, the protein had about half of its original activity. But the Ca++ complex of this derivative was almost fully active. Carbamylation of the remaining two amino groups inactivated DNase and Ca++ did not restore activity; therefore, the positive charges on these groups probably help to maintain the active structure of the enzyme. It is not likely that the two amino groups are involved in binding Mn++-DNA, since they do not seem to be freely available for reaction with cyanate. Trinitrophenylation gave results similar to those of carbamylation, but only one amino group of Ca++-free DNase or four to five groups of Ca++-DNase could be trinitrophenylated without inactivation. The neutral, hydrophobic trinitrophenyl groups probably distort the enzyme more than carbamyl groups do. Two to three amino groups resisted trinitrophenylation, which may also indicate that these groups have structural roles. Simplified procedures are described for the chromatographic determination of homocitrulline and for the spectrophotometric titration of free amino groups with 2,4,6-trinitrobenzenesulfonic acid. The α-picolinimidyl, e-guanidino derivative of DNase A was active, and its metal-chelating picolinimidyl group could facilitate the preparation of isomorphous derivatives for x-ray studies.

Homocitrulline was found to be normally present in the urine of infants and young children up to the age of 6 years, in amounts varying from 1 to 65 mg./24 hr. With few exceptions, the amount found in the urine of adults was too low to be estimated accurately. It was not present in milk or milk products. Homoarginine could not be detected in urine.