Enzymatic Hydrolysis Method Research Articles

Amides are novel protein modifications formed by physiological sugars

The elucidation of structure and formation of AGEs is of major interest to understand related pathophysiologies, such as the late complications in diabetes, atherosclerosis and aging, and to develop pharmaceutical strategies for their prevention [1,2]. The discrepancy between extent of protein modification and of fluorescence has led to the notion that the major sugar-derived protein modifications cannot be explained by colored, UV and fluorescence active structures. Furthermore, evidence has shown that potential structures must be labile to acid and alkaline hydrolysis and cannot be stabilized by sodium borohydride reduction [3].Following this concept, N6-{2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl}lysine (GOLA) and N6-glycoloyllysine (GALA) were identified as novel amide protein modifications mediated by physiological reducing sugars [4]. Their identity was unequivocally confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with Nα-t-BOC-lysine revealed glyoxal and glycolaldehyde as immediate precursors with reaction pathways directly linked to Nε-carboxymethyllysine (CML) via glyoxal–imine structures. Based on a common intermediate, the ratio of the amide crosslink GOLA to the imidazolium crosslink GOLD was favored towards the amide at lower glyoxal levels in incubations conducted at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose with lysine was directly dependent on oxidation. Presence of aminoguanidine almost completely suppressed GALA formation, whereas GOLA was only inhibited by 16%. As the latter effect was exactly in line with changes in CML synthesis, it can be reasoned that during Amadori product degradation, 84% of GOLA originates via direct oxidative fragmentation and 16% via glyoxal–imine intermediates. In contrast, the formation of GALA must be explained solely via glyoxal–imines. An efficient enzymatic hydrolysis and analytical method was developed for the detection of labile amide modifications in highly modified proteins and in vivo. In a first attempt, GOLA was quantified in brunescent lens protein at 66 pmol/mg protein. This suggested that amide modifications are of major physiological relevance in comparison to the already established AGEs pentosidine, GOLD and MOLD.

Determination of amino acids in twoPolysiphonia species and study of enzymatic hydrolysis method

The total content of the rich amino acids in two common red algae,Polysiphonia urceolata andPolysiphonia japonica growing in the Qingdao seashore were determined. The algae powder was hydrolyzed by 6 mol/L HCl at 110°C for 48 h and determined by amino acid analyzer. The content was 25.35% and 24.16%, respectively, much higher than that of some other species. In addition, a nutritive liquid with abundant amino acids was prepared (by the enzymatic hydrolysis method usingPolysiphonia urceolata) as raw material for a kind of health beverage. The dried seaweed was decolored by 0.25% KMnO4 and 0.5% active carbon, then enzymalized. In the selection of enzymalizing condition, the orthogonal experimental design was used. Four factors including kinds of enzyme, quantity of enzyme, temperature and time were studied at 3 levels. According to the orthogonal design results, we can choose an optimal condition: hydrolyzing at 45°C by neutral proteinase (0.25%, w/w) for 2h, adjusting pH to 8.5, then adding trypsin (0.25%, w/w) and hydrolyzing for 2 h. Finally the above solution was alkalized by NaOH and neutralized by casein. After the hydrolyzed liquid was filtered and concentrated, suitable additives were added. The final products contain rich amino acids.

Identification of Nω-carboxymethylarginine as a novel acid-labile advanced glycation end product in collagen

Collagen undergoes continuous non-enzymatic glycation during its long life period. The products resulting from the glycation reaction, so-called advanced glycation end products (AGEs), were regarded as potential pathogens of various diseases such as diabetic complications. Although several AGEs were identified from acid hydrolysates of glycated collagen, the major AGE(s) responsible for the diseases have not yet been fully characterized. Moreover, acid-labile constituents were decomposed during acid hydrolysis. To investigate these AGEs, we used the enzymatic hydrolysis method [Bensusan, Dixit and McKnight (1971) Biochim. Biophys. Acta 251, 100-108]. As a result, an acid-labile unknown compound was discovered from the digested glycated collagen. We identified this compound as NΩ-carboxymethylarginine (CMA) by matrix-assisted laster-desorption ionization-MS and NMR. CMA gradually increased in collagen during incubation with glucose and the yield reached about 8 mol/mol of collagen, which is 100 times higher than that of pentosidine. This result suggests that CMA is a major AGE in collagen.

Identification of Nω-carboxymethylarginine as a novel acid-labile advanced glycation end product in collagen

Collagen undergoes continuous non-enzymatic glycation during its long life period. The products resulting from the glycation reaction, so-called advanced glycation end products (AGEs), were regarded as potential pathogens of various diseases such as diabetic complications. Although several AGEs were identified from acid hydrolysates of glycated collagen, the major AGE(s) responsible for the diseases have not yet been fully characterized. Moreover, acid-labile constituents were decomposed during acid hydrolysis. To investigate these AGEs, we used the enzymatic hydrolysis method [Bensusan, Dixit and McKnight (1971) Biochim. Biophys. Acta 251, 100-108]. As a result, an acid-labile unknown compound was discovered from the digested glycated collagen. We identified this compound as N(omega)-carboxymethylarginine (CMA) by matrix-assisted laster-desorption ionization-MS and NMR. CMA gradually increased in collagen during incubation with glucose and the yield reached about 8 mol/mol of collagen, which is 100 times higher than that of pentosidine. This result suggests that CMA is a major AGE in collagen.

Enzymatic hydrolysis, extraction, and quantitation of retinol and major carotenoids in mature human milk

An improved method for enzymatic hydrolysis, extraction, and high performance liquid chromatography (HPLC) separation of major carotenoids and retinol of mature human milk is described. After pretreatment with bile salts and protease, carotenoids and retinol are released by lipase treatment followed with brief chemical saponification. The HPLC method provides enhanced peak resolution and improved elution profiles. The procedure is nondestructive to provitamin-A carotenoids while effectively hydrolyzing retinyl esters. Using this method, we were able to significantly increase the recovery of both β-carotene and retinol from human milk over previously published procedures. The method is sensitive to picomolar quantities of carotenoids and retinol. Retinol, lutein/zeaxanthin, β-cryptoxanthin, lycopene, α-carotene, and β-carotene are effectively recovered and quantitated from a single 1-mL sample of milk.

A modified method to minimise losses of carotenoids and tocopherols during HPLC analysis of red palm oil

A well-established saponification method for red palm oil was compared with a newly developed enzymatic hydrolysis method followed by analysis using highperformance liquid chromatography. Applying the saponification method to extract carotenoids from red palm oil resulted in soap precipitation and significant losses of α- and β-carotene ( P < 0.01) and α-tocopherol ( P < 0.05). The process also facilitated the isomerisation of α-carotene and β-carotene ( P < 0.05). Using the non-specific Candida cylindracea lipase, complete hydrolysis of red palm oil was achieved after 4 h and no significant loss of carotenoids and tocopherols occurred during the extraction procedure. Mild hydrolysis with the use of Candida cylindracea lipase enables quantitative analysis of carotenoids and tocopherols in red palm oil without isomerisation or destruction of either carotenoids or tocopherols. The present method is comparable or shorter in time than the conventional saponification method.

Nuclear magnetic resonance spectroscopic characterization of carboxymethylcellulose

AbstractPrevious assignment of the signals in the spectra of carboxymethylcellulose, based on an incremental calculation and hence on low‐molar‐mass model substances, made determination of the partial degree of substitution fundamentally impossible because overlapping of the relevant signals was predicted. However, the methods of acid and enzymatic hydrolysis employed generate the monomer or a mixture of polymers, oligomers and monomers and hence a large number of end groups, which also influence the nuclear magnetic resonance (NMR) spectra. By using ultrasound, it was possible to degrade the molar mass (lower limit of molar mass approx. 100000 g/mol), without oligomers being generated and cleavage of side groups occuring. The viscosity of 10 wt.‐% polymer solutions was so low that the partial degree of substitution could be determined quantitatively for the first time. An additional 13C NMR spectroscopic examination of the acid hydrolysate enabled the composition of the eight monomers of sodium carboxymethylcellulose (NaCMC) to be determined in a degree of substitution (DS) range of 0,8–3,0. Knowledge of the monomer compositions is then used to reassign the signals of the polymer spectra, which are then evaluated. Comparison with titrimetric methods (polyelectrolyte titration and ASTM method) showed some wide discrepancies with the absolute method (NMR) in the case of samples with a DS greater than 1. The results of the 13C NMR spectroscopic examination of the acid hydrolysate were also used to determine the relative rate constants of the etherification reaction (k2:k3:k6 = 3,0:1,0:2,1), and these were then compared with published data.

Protein digestibility of extruded cereal grains

Seven whole-grain cereals were examined to determine extrusion processing effects on in-vitro protein digestibility. Eight extrusion conditions were applied and effects of cereal variety, extrusion temperature, RPM and feed moisture on in-vitro protein digestibility were studied. Protein digestive acceptability of unprocessed and extruded cereals was determined using an in-vitro method of enzymatic hydrolysis. The most protein-digestible products were produced at extrusion combination: 15% feed moisture, 100°C/150°C product temperature and 100 rpm. This effect was significant when considered across all cereals. Results of this study suggest that a particular combination of extrusion process conditions may be applied when extruding a wide variety of cereals, with the benefit of improving protein digestibility.

31] High resolution analysis of carotenoids in human plasma by high-performance liquid chromatography

Publisher Summary This chapter describes chromatographic separation, which is applicable to the analyses of tissue samples. Many human tissues contain negligible lipid other than cholesterol and phospholipid, and these lipids cause minimal interference with the procedure of chromatographic separation. The enzymatic hydrolysis method is suitable for elimination of neutral lipids in plasma samples, but in future, enzymatic hydrolysis may be adapted to eliminate the bulk triglyceride found in adipose tissue and milk. The chapter discusses procedures, which have wide applicability to a variety of carotenoid analysis methods. Future developments in high-performance liquid chromatography (HPLC) column technology may allow of high-resolution separation to be carried out in a shorter time period. However, if this HPLC procedure were adapted to an autosampler, it might be practical to use the method to analyze the large number of samples evaluated in typical epidemiological investigations.

Measurement of Conjugated Catecholamines in Human Plasma. A Low Cost HPLC-ECD Method

Abstract We present an HPLC assay with electrochemical detection of conjugated catecholamine levels in human plasma. This method allows the direct assessment of the conjugated catecholamine values without the subtraction of the free catecholamine amounts: the assay is performed in the residual supernatant after the extraction (and the assay) of free catecholamines. The results are comparable with the classic enzymatic and acid-neat hydrolysis methods (which yield the sum of free and conjugated catecholamine concentrations). This technique shows good reproducibility (within-run and between-run CVs < 8% in the physiological range), and sensitivity (< 20 pg per each deconjugated catecholamine per extract). This method allows about 30 low cost determinations of plasma free and conjugated catecholamines to be done in a working day.

Synthesis of oligodeoxyribonucleotides containing 4-N-(ω-aminohexyl)-5-methyldeoxycytidine

Oligodeoxyribonucleotides containing 4-N-(ω-aminohexyl)-5-methyldeoxycytidine have been synthesized. It has been shown that a method of preparation based on the use of a monomeric component containing 4-N-(ω-aminohexyl)-5-methyldeoxycytidine is more reliable and promising than transamination at the level of an oligonucleotide containing 5-methyl-4-triazolyl-6-deoxythymidine. A structure of the monomeric component of oligonucleotide synthesis bearing a hexylamine grouping with trifluoroacetyl protection in the aliphatic amino group has been put forward. The structure of the modified deoxyribonucleotides obtained was confirmed not only by the method of chemical modification, degradation, and exhaustive enzymatic hydrolysis but also by obtaining derivatives at the aliphatic amino group with fluorescein isothiocyanate.

Effect of Maillard Browning Reaction on Protein Utilization and Plasma Amino Acid Response by Rainbow Trout (Salmo gairdneri)

The effect of the Maillard browning reaction in the diet of rainbow trout (Salmo gairdneri) on growth and amino acid availability was investigated. Chemical and enzymatic hydrolysis methods were applied for the detection of the losses of amino acids in a model protein browning system. Arginine and lysine exhibited the greatest losses in the mixture of fish protein isolate and glucose stored for 40 d at 37°C. The apparent digestibility and absorption of individual amino acids, particularly lysine, was lower in trout fed browned protein than in those fed the control protein. Plasma lysine levels were significantly depressed, while the plasma levels of glucose and most other amino acids were elevated in relation to the loss in nutritive value of dietary protein after browning. The early Maillard reaction derivative of lysine, ε-deoxy-fructosyl-lysine, was recovered from browned protein (by using the in vitro enzymatic hydrolysis procedure) and from the plasma of trout fed browned protein. Analysis of plasma free amino acids provided an indication of lysine bioavailability and identified lysine as the first-limiting amino acid in the diets containing browned protein.

Polysaccharides from the inflorescences ofMatricaria matricarioides

Fractionation of the polysacchride complex of the inflorescences of pineapple weed has given a galacturonan and a pectic acid. The structures of the galacturonan and pectic acid have been studied by the methods of enzymatic hydrolysis, periodate oxidation, and exhaustive methylation. It has been established that the galacturonan is a linear polysaccharide consisting of D-galacturonic acid residues in the pyranose form with α-1→4-bonds. The main polysaccharide chain of the pectic acid consists of D-galacturonic acid residues in the pyranose form, D-galactose, L-arbinose, and D-xylose residues are covalently bound to carbon atoms 2 or 3 of the main chain of the polysaccharide.

でん粉に関連した食品用低分子糖質

Recently, we have seen remarkable progress in the enzymology and in the technological applications of enzymes to starch industry. By the end of 1960, the dextrose production of Japan was swiftly switched from the acid hydrolysis to the enzymatic hydrolysis method. Besides dextrose and syrup, at present, isomerized syrups, maltitol, cyclodextrins, coupling sugar and maltooligosaccharides are utilized and employed for other many purposes. Present pictures of starch hydrolyzing industry in Japan are mentioned in this paper.

Enzymatic deconjugation of catecholamines in human and rat plasma and red blood cell lysate

We have developed a method for enzymatic hydrolysis of both sulfated and glucuronidated catecholamines in plasma and red blood cell lysate. Hydrolysis occurs in the course of the radioenzymatic assay for catecholamines. In human plasma, catecholamines are conjugated almost entirely with sulfate while, in rat plasma, glucuronides are the main conjugates of epinephrine and dopamine but not norepinephrine. Rat plasma contains less percent conjugated catecholamine than human plasma. Human red blood cell lysate contains less conjugated catecholamine than plasma, whereas free E in lysate exceeds that of plasma and free NE has same level both in plasma and lysate. This method is useful in detecting total (free + sulfated + glucuronidated) catecholamines and the nature of conjugated catecholamines.

Efficient enzymatic hydrolysis of polyprenyl pyrophosphates

A method of efficient enzymatic hydrolysis of polyprenyl pyrophosphates was established. Polyprenyl pyrophosphates such as all- trans-octaprenyl and all- trans-nonaprenyl (solanesyl) pyrophosphate can be hydrolyzed completely to the corresponding primary alcohols by the action of potato acid phosphatase in the presence of 60% (v/v) methanol.

Quantitative enzymatic hydrolysis of tRNAs : Reversed-phase high-performance liquid chromatography of tRNA nucleosides

A rapid quantitative method for enzymatic hydrolysis of microgram amounts of tRNA has been developed, specifically to take full advantage of our precise, accurate, and selective reversed-phase high-performance liquid chromatographic (HPLC) system for separation and measurement of the major and modified nucleosides in tRNA. After study of several enzyme systems, nuclease P1 and bacterial alkaline phosphatase were selected and the hydrolysis parameters were systematically studied. Optimized hydrolysis conditions give quantitative hydrolysis in 2 h and this short incubation time prevents loss of unstable nucleosides. The chromatographic system can tolerate relatively high levels of protein in the sample allowing high enzyme—substrate ratios and direct injection of hydrolysates. This enzymatic hydrolysis—HPLC method is the best described to date for quantitative determination of the nucleoside composition of tRNAs and has already provided important information for investigation of the role of modification in the function of RNAs.

Studies on the measurements of urinary pregnanediol by radioimmunoassay (author's transl)

A simple and practical radioimmunoassy (RIA) for pregnanediol without any pretreatment of urine specimens such as hydrolysis and/or extraction was developed. Antisera were produced by immunizing rabbits with pregnanediol-3-glucuronide (P-diol-3-G)-BSA conjugates and P-diol-3-hemisuccinate-BSA conjugates. The specificities of the antisera were examined by their cross-reactivities with related steroids. Antiserum to P-diol-3-G-BSA was significantly cross-reacted with 20 alpha-OH-P(201%), P-diol(100%), P -diol-3-G (77.3%, where 119% as free P-diol), to a lesser extent with 20 beta-OH-P(17.8%), and did not react with other steroids. Antiserum to P-diol-3-hemisuccinate-BSA, on the other hand, was capable of reacting with P-diol(100%), P-diol-3-G(46.9%), 20 alpha-OH-P(75.2%) and 20beta-OH-P (8.97%), respectively. Antiserum generated against P-diol-3-G-BSA was almost twice as high in its avidity for P-diol-3-G as compared with antiserum raised against P-diol-3-hemisuccinate-BSA. Antiserum to 20alpha-OH-P-3-carboxy-methyl oxime-BSA conjugate was only reactive with 20alpha-OH-P and not with P-diol or P-diol-3-G. From the data presented here, antiserum to P-diol-3-G-BSA with high specificity toward urinary P-diol-3-G was used for the present assays. Ammonium sulfate precipitation was employed for the separation of antibody-bound and free tracer hormones. A direct method in which simply diluted urine was directly applied to RIA and the enzymatic hydrolysis method which requires extraction of P-diol with ether following hydrolysis of urine by beta-glucuronidase were both examined in the present RIA on the same specimens which contained known amounts of 3H-P-diol-3-G. Values obtained by the direct method were in good correlation with those determined by the hydrolysis method (r=0.994 p less than 0.01, Y=1.07X + 0.30). The working range of the assay was from 50pg to 5ng of P-diol per tube, and intra- and interassay precisions were 9.8, 12.5 and 12.9, 16.7%, respectively. Determination of radioimmunoassayable P-diol-3-G in urine was also in good correlation with the values obtained by the currently used analytical procedure, the Klopper-Kambegawa method.

Preparation of prostaglandin E2 from Plexaura homomalla

An efficient method for enzymatic hydrolysis of esters of PGA2 contained in the sea whip, Plexaura homomalla(var.S) and subsequent conversion of the released PGA2 to PGE2via silylation, epoxidation, and reductive opening of the epoxide is described.

The Specific Distribution of Fatty Acids in the Glycerides of Animal and Vegetable Fats

The structure of naturally occurring triglycerides has been studied over a number of years with little agreement about the distribution of the fatty acids, except as to the prevalence of mixed glycerides. Several types of arrangement, for example, even, random, partial random, and restricted random, have been proposed and defended. Hilditch (I) and Bhattacharyya (2) have recently reviewed the bases of these various theories. One reason that a number of theories developed has been the lack of suitable methods for the isolation and characterization of glycerides. It is possible, with fairly quantitative yields, to separate the triglycerides of a natural fat into glyceride classes, i.e. trisaturated, disaturated, monosaturated, and triunsaturated. However, only with a limited number of fats has it been possible to separate major proportions of single glycerides which can be identified by thermal and x-ray diffraction techniques. For instance, it has been shown that 2-oleoyl distearin comprises approximately 60 per cent of kokum butter (3, 4), that 2-oleoyl dipalmitin is the principal single triglyceride of stillingia tallow (5), and that 2-oleoyl palmitoyl stearin is present to the extent of 50 per cent in cocoa butter (6-8). Thus, the conclusively identified disaturated glycerides of vegetable oils are exclusively 2-oleoyl disaturated. On the other hand, lard has a very high proportion of 2-palmitoyl stearoyl “olein,” clearly in contrast with beef and mutton tallow (9, 10). Thus, the evidence, although scattered, is indisputable for the occurrence in several different organisms of specific glycerides in proportions far in excess of expectations for random distribution. Nevertheless, reports continue to appear containing data which are interpreted as supporting an element of randomness in glyceride organization (11, 12). In 1956 Mattson and Beck (13) demonstrated the specificity of pancreatic lipase for the cleavage of ester linkages at the 1 and 3 positions of triglycerides and suggested that this offered a new tool for study of triglyceride structure, a tool with certain obvious advantages over previous means. This method of enzymatic hydrolysis has been applied by Savary, Flanzy, and Desnuelle (7) to fractions of eight natural glycerides. On the basis of iodine value and, in some instances, identification of the fatty acids, they concluded that the fatty acids were not distributed at random. In the present study the enzymatic technique, applied to twelve vegetable fats and seven animal fats, has yielded strong evidence for a high degree of specificity in fatty acid distribution in the glycerides of natural fats.