Aspergillus Oryzae Α-amylase Research Articles

Evaluation of Porcine and Aspergillus oryzae α-Amylases as Possible Model for the Human Enzyme

α-amylases are ubiquitous enzymes belonging to the glycosyl hydrolase (GH13) family, whose members share a high degree of sequence identity, even between distant organisms. To understand the determinants of catalytic activity of α-amylases throughout evolution, and to investigate the use of homologous enzymes as a model for the human one, we compared human salivary α-amylase, Aspergillus oryzae α-amylase and pancreatic porcine α-amylase, using a combination of in vitro and in silico approaches. Enzyme sequences were aligned, and structures superposed, whereas kinetics were spectroscopically studied by using commercial synthetic substrates. These three enzymes show strikingly different activities, specifically mediated by different ions, despite relevant structural homology. Our study confirms that the function of α-amylases throughout evolution has considerably diverged, although key structural determinants, such as the catalytic triad and the calcium-binding pocket, have been retained. These functional differences need to be carefully considered when α-amylases, from different organisms, are used as a model for the human enzymes. In this frame, particular focus is needed for the setup of proper experimental conditions.

The Inhibitory Effect of Hydroalcoholic Extract from the Algerian Echium trygorrhizum Pomel Roots on α-amylase Activity

Background: Species of Echium trygorrhizum Pomel are used traditionally in Algeria folk medicine for the treatment of Diabetes, Jaundice and Tonsillitis. To our knowledge, no previous study has been conducted on the pharmacological activities of this species. Objective: The objective of the present research was to evaluate the content of polyphenols, flavonoids and condensed tannins compounds and to assess in vitro the antioxidant activity and the inhibitory effect of the hydroalcoholic extract of this plant on α-amylase activity, an enzyme responsible for digestion of carbohydrate before the process of intestinal absorption. Methods: Polyphenols, flavonoids and condensed tannins were evaluated spectrophotometrically using Folin-Ciocalteu, the Aluminum chloride and vanillin methods, respectively. The antioxidant activity using DPPH radical scavenging, ABTS, Ferric reducing antioxidant power and β-carotene bleaching tests and the assessment of in vitro α-amylase inhibitory potential by an Aspergillus oryzae α- amylase have been studied. Results: The hydroalcoholic crude extract was able to inhibit the α-amylase enzyme in vitro, with an IC50 of 0.56 ± 0.044 mg/ml, in addition, the contents of polyphenols and flavonoids were found to be 157.403 ± 0.835 μg GAE/mg extract and 30.156±2.634μg Q E / mg extract, respectively, Whereas the amount of tannins was 65.293 ± 0.883μg Cat E/ mg of dry extract. Conclusion: The present study revealed that the extract is rich in phenolic compounds, which play a really important role in the scavenging of free radicals. The inhibitory capacity of hydroalcoholic roots extract on Aspergillus oryzae α-amylase enzyme has been reported and might be used as a natural agent within the management of diabetes mellitus.

Structures of two novel crystal forms of Aspergillus oryzae alpha amylase (taka-amylase)

The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65Å and 1.43Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (β/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A.oryzae enzyme model, which predicts substrate interacting amino acid residues.

Aspergillus oryzae α-amylase supplementation on rumen volatile fatty acid profile and relative abundance of mRNA associated with nutrient absorption in ruminal and duodenal tissue from beef steers

ABSTRACT The objective of this experiment was to investigate the effect of Aspergillus oryzae α-amylase (AAM) supplementation on rumen VFA profile and relative abundance of mRNA associated with nutrient absorption in ruminal and duodenal tissue from beef steers. Nine crossbred beef steers (BW 622 ± 50 kg), fitted with rumen and duodenal fistulas, were used in this experiment. Steers were housed in individual stanchions and fed a high-concentrate finishing diet (6% forage NDF) twice daily for 8 d. Treatments included (1) control (corn meal; n = 5) and (2) AAM (fungal α-amylase 750 units/g; n = 4). Dietary treatment supplements were manufactured before each feeding by mixing 3 g of α-amylase or corn meal into 150 g of dried distillers grains for the morning feeding and 2 g of α-amylase or corn meal into 100 g of dried distillers grains for the afternoon feeding. Supplements were applied as a top dress for every feeding and thoroughly mixed by hand. On d 5, rumen fluid samples were obtained every 4 h for 24 h and analyzed for VFA. On d 9, rumen papillae and duodenal mucosal tissue samples were collected. Total tissue RNA was extracted for real-time PCR analysis. Sodium/potassium ATPase pump α1; glucose transporter 2 and 5; putative anion transporter isoform 1; sodium/hydrogen antiporter isoforms 1, 2, and 3; 3-hydroxy 3-methylglutaryl coenzyme A synthase isoform 2; downregulated in adenoma; monocarboxylate cotransporter isoform 1; and glyceraldehyde-3-phosphate dehydrogenase mRNA were tested. No treatment differences (P > 0.21) were detected for any of the genes analyzed in ruminal or duodenal tissue. Concentrations of VFA and the acetate-to-propionate ratio did not differ (P > 0.22) among treatments. However, the acetate-to-propionate ratio and molar percentages of butyrate were numerically greater (P = 0.17) in AAM steers compared with controls. Under the conditions of this experiment, AAM supplementation had no effect on relative expression of mRNA associated with nutrient absorption and minimal effects on molar proportions of VFA.

α-Amylase inhibitory effect of 3β-olean-12-en-3-yl (9Z)-hexadec-9-enoate isolated from Spondias mombin leaf

Spondias mombin is a traditional herb used in the treatment of diabetes mellitus by traditional healers in southwest Nigeria. In this study, we investigated the antidiabetic activity using α-amylase inhibitory assay and isolated an active compound. The bioactivity assay-guided study demonstrated the presence of an α-amylase inhibitory fraction from S. mombin leaf. An active compound, 3β-olean-12-en-3-yl (9Z)-hexadec-9-enoate was also studied. This is reported, from this plant, for the first time. The methanol extract, diethyl ether fraction and the isolated compound exhibited significant enzyme inhibitory activity against Aspergillus oryzae α-amylase. Our study revealed, for the first time, the isolation and α-amylase inhibitory activity of 3β-olean-12-en-3-yl (9Z)-hexadec-9-enoate from S. mombin leaf.

Comparative studies of two α-amylases acting on two Sorghum hybrids starches (Montecillos hybrid 2 and 3) and their significant differences in their catalytic activities

Starchy substances constitute the major part of the human diet for most people in the world. α-Amylases are widely distributed enzymes that initiate the hydrolysis of starch into low molecular weight maltodextrins. The effect of pH on the rate of hydrolysis of both sorghum (Montecillo 2 and 3) hybrids starches by industrial bacterial amylase (IBA) and Aspergillus oryzae α-amylase(AOA) was studied. This result suggests that both IBA and AOA α-amylases had slightly high affinity for soluble starch of sorghum Montecillo 3 and 1 hybrids, respectively. Using the structural information available, the theoretical p K a values of its ionizable residues directly involved in the catalytic region were determined. Both our experimental data and prediction studies indicated that the average p K a values are in good agreement.

Dietary supplementation of ruminant diets with an Aspergillus oryzae α-amylase

Research in the area of dietary enzyme supplements for ruminant diets has primarily focused on fibrolytic enzymes, while activities involved in the process of starch digestion have been largely ignored. Since starch represents a major component in diets fed to highly productive cattle, the use of enzymatic dietary supplements to manipulate starch digestion in the rumen may allow improved productivity. This review discusses current information on dietary supplementation of calf, dairy and beef cattle diets with an Aspergillus oryzae extract containing α-amylase activity. During starch hydrolysis, α-amylase randomly cleaves starch polymers to low molecular weight oligosaccharides and eventually produces maltotriose and maltose from amylose and α-limit dextrins, maltose and glucose from amylopectin. Through its hydrolytic action, supplemental α-amylase hypothetically increases the availability of starch hydrolysis products in the rumen consequently altering the ruminal fermentation process. Data from studies employing lactating dairy cows, steers or rumen-simulating continuous cultures suggest that supplemental α-amylase did not increase ruminal starch digestion but consistently increased butyrate and reduced propionate molar proportions in the rumen. The increase in ruminal butyrate was also reflected in higher blood β-hydroxybutyrate concentrations in both transition and lactating dairy cows. In addition, supplemental α-amylase enhanced ruminal epithelium growth in dairy calves, a tissue that preferentially uses butyrate as an energy source. Experiments with pure cultures of ruminal bacteria showed that supplemental α-amylase supported rapid growth of bacteria that cannot grow, or grow slowly, on starch such as Butyrivibrio fibrisolvens D1, Selenomonas ruminantium GA192 or Megasphaera elsdenii T81. In contrast, bacteria that grow rapidly on starch, such as Streptococcus bovis S1 or Butyrivibrio fibrisolvens 49, did not benefit from α-amylase supplementation. Animal performance studies showed higher weight gain, and longissimus muscle area, in finishing beef cattle fed supplemental α-amylase. Weight gain improvements were primarily mediated through increased dry matter intake, which may be a consequence of reduced ruminal propionate molar proportions reported in other studies. In lactating dairy cattle, supplemental α-amylase increased milk yield, reduced milk fat proportion without reducing milk fat yield and tended to improve milk protein yield when data from 45 commercial herds (approximately 8150 cows) were examined. Currently available data on effects of the Aspergillus oryzae α-amylase described here suggest that this enzyme supplement may improve animal productivity by modifying ruminal starch digestion without necessarily increasing starch digestion in the rumen.

α-Amylase-catalysed synthesis of alkyl glycosides

Alkyl glycosides were synthesised from starch and alcohols using Aspergillus oryzae α-amylase as catalyst. In the degradation of starch by α-amylase, the alcohols competed with water as glycosyl acceptors. In the reaction with methanol, methyl maltoside and methyl maltotrioside were the main alcoholysis products. Conversion of 45 g/l starch in 30% methanol resulted in a product mixture containing 26 mM maltooligosaccharides and 3.6 mM methyl glycosides. With ethanol, propanol and butanol, alkyl maltosides and alkyl maltotetraosides were detected, and with benzyl alcohol, benzyl glycosides having two, three or five glucose units were formed. No alcoholysis reaction occurred with hexanol or octanol. In conclusion, α-amylase is promising for the one-step synthesis of alkyl glycosides having more than one monosaccharide unit, which are difficult to synthesise in other ways.

Activation and stabilization of 10 starch-degrading enzymes by Triton X-100, polyethylene glycols, and polyvinyl alcohols

A dilute solution (0.01–0.1 unit/mL) of porcine pancreatic α-amylase (PPA) at pH 6.5, 24 °C, and 1 mM CaCl 2 was found to lose 98% of its activity on standing for 2 h. Addition of 0.02% (w/v) Triton X-100 gave 41% activation and stabilization for 3 h. Because Triton X-100 has a polyethylene glycol side chain, seven polyethylene glycols (PEGs), ranging in MWs of 400–8000 Da and two polyvinyl alcohols (PVAs) of MWs of 10,000 and 50,000 Da were tested as activators and stabilizers of 10 starch-degrading enzymes: PPA, human salivary α-amylase (HSA), Aspergillus oryzae α-amylase (AOA), Bacillus amyloliquefaciens α-amylase (BAA), Bacillus licheniformis α-amylase (BLA), Aspergillus niger glucoamylase (GA), barley β-amylase (β-A), Pseudomonas amylodermosa isoamylase (IA), Bacillus acidopullulyticus pullulanase (PUL), and Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase). Although nearly all of the additives gave activation and stabilization of the 10 enzymes, there was one specific additive and concentration for each enzyme that gave a maximum degree of activation: 0.02% (w/v) PEG 1500 Da gave 77% activation for β-A; 0.04% PEG 1500 Da gave 70% for PPA; 0.04% PEG 2000 Da gave 58% for IA; 0.04% PEG 1500 Da gave 53% for AOA; 0.04% PVA 50,000 Da gave 48% for GA; 0.04% PEG 4500 Da gave 46% for BLA; 0.02% Triton X-100 gave 45% for HSA; 0.04% PEG 1000 Da gave 42% for BAA; 0.02% Triton X-100 gave 27% for PUL; and 0.02% PEG 1500 Da gave 20% for CGTase. The mechanism of activation and stabilization is postulated to be the binding of the additives to the enzyme-proteins to give a single tertiary structure that results in a maximum enzyme activity, whereas without the additive the enzymes have several tertiary structures in equilibrium, each with a specific activity that results in an average lower activity.

Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae α-amylase

The physiology of three strains of Aspergillus nidulans was examined—a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae α-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and α-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the α-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of α-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of α-amylase expression. The effect of different carbon sources on the α-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l −1 of α-amylase was reached after 162 h of cultivation.

Evaluation of different promoters for the efficient production of heterologous proteins in baker's yeast

We have constructed reporter gene expression vectors placing the Aspergillus oryzae α-amylase cDNA and the Geotrichum candidum lipase 2 gene, under the control of the yeast gene promoters,ACT1, ADH1, PGK1, and TDH1. Expression regulated by the shorted form of the ADH1 promoter gave the highest proteins yield for cells cultured in molasses medium or flour/water mixtures. Nevertheless, the ACT1 promoter appeared as the strongest in cells growing actively with sucrose.

Structural consequences of neopullulanase mutations

Bacillus stearothermophilus neopullulanase (NPL) structure was modeled based on Aspergillus oryzae α-amylase (TAA) to understand the structure-function relationships of this pullulan hydrolyzing enzyme. The NPL structure seems to consist of a central (α/β) 8 barrel to which the other domains are attached. The immediate surroundings of the NPL catalytic site were found to have very similar structure to TAA. The more distant sites are different due to the stereochemical requirements of accommodating in the substrate α-1,6-linkages at every third position instead of α-1,4-linkages. The substrate binding cleft is wider than in α-amylases. The NPL structure, function, substrate binding and the consequences of mutations were discussed based on the modeled structure.

Close evolutionary relatedness among functionally distantly related members of the ( α/ β) 8-barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region

A short conserved sequence equivalent to the fifth conserved sequence region of α-amylases (173_LPDLD, Aspergillus oryzae α-amylase) comprising the calcium-ligand aspartate, Asp-175, was identified in the amino acid sequences of several members of the family of ( α/ β) 8-barrel glycosyl hydrolases. Despite the fact that the aspartate is not invariantly conserved, the stretch can be easily recognised in all sequences to be positioned 26–28 amino acid residues in front of the well-known catalytic aspartate (Asp-206, A. oryzae α-amylase) located in the β4-strand of the barrel. The identification of this region revealed remarkable similarities between some α-amylases (those from Bacillus megaterium, Bacillus subtilis and Dictyoglomus thermophilum) on the one hand and several different enzyme specificities (such as oligo-1,6-glucosidase, amylomaltase and neopullulanase, respectively) on the other hand. The most interesting example was offered by B. subtilis α-amylase and potato amylomaltase with the regions LYDWN and LYDWK, respectively. These observations support the idea that all members of the family of glycosyl hydrolases adopting the structure of the α-amylase-type ( α/ β) 8-barrel are mutually closely related and the strict evolutionary borders separating the individual enzyme specificities can be hardly defined.

Expression of Aspergillus oryzae α-amylase gene in Saccharomyces cerevisiae

A fragment containing the full length cDNA from Aspergillus oryzae α-amylase has been amplified by PCR using specific synthetic oligonucleotides. The amplified cDNA was designed to favour its expression in yeast by modifying its upstream untranslated region. It was subcloned in the expression vector pYEXα1, placed under the control of the yeast CYC1-GAL10 promoter and used to transform Saccharomyces cerevisiae. Cells were then able to express and secrete active α-amylase to the medium in a regulated fashion. The recombinant enzyme had similar electrophoretic mobility and catalytic properties to the original A. oryzae α-amylase.

サイクロデキストリンの基礎と応用に関する研究

1. Cyclodextrin-producing enzyme produced by Bacillus macerans (CGTase, macerans enzyme) Crude macerans enzyme preparation was purified and the enzyme was crystallized. Elucidation of the CDs' forming path : By use of various kinds of substrates, well-known phenomenon of p-CD accumulation at final stage of the reaction was explained from time course data of CD formation and the rate of coupling. Elucidation of cyclizing reaction : Cyclizing reaction proceeds from non-reducing ends of the substrates cyclizing 6 glucose units to form α-CD and the remaining fragments. The rate of cyclization was high on substrates having more than 8 glucose units, and accelerated by the addition of helices forming reagents ; moreover, it was found that α-and β-CDs were selectively formed by the addition of 65 and 76 helices forming reagents. Branched CDs were produced by forming helical structure with surface active reagents from substrates having branches, and in the same manner, 1 or 2 hydroxyethyl substituted CDs were produced by the action of macerans enzyme on the helix formed hydroxyethyl starch.Elucidation of coupling action : Glucosyl-α-CD and 14C-labeled glucose were reacted with macerans enzyme, and the structure of the formed branched oligosaccharides was analyzed. The smallest non-radioactive branched oligosaccharide was B4(63-α-glucosylmaltotriose), and at the initial stage of the reaction, radioactive(*) Bs(mainly 66-α-glucosylmaltoheptaose)was formed to be gradually degraded to B7* (mainly 65-α-glucosylmaltohexaose), B6 (mainly 64-α-glucosylmaltopentaose) and B5 (64-α-glucosylmaltotetraose) From the results described above, the author proposed an enzyme model of active site.2. Production of CDs Methods for the production of α-CD, α-CD containing starch syrup, γ-CD were developed, and a large ringed CDs preparation method, by which δ-θ-CDs were produced, was also developed. δ-θ-CDs preparation contained a fair amount of intra branched CDs which have α-1, 6 linkage in the CDs themselves. 3. Production and the properties of branched CDs A method for the production of branched CDs which have branch(es) longer than glucosyl unit was developed, and maltosyl-CDs (G2-CDs) were effectively produced. The number of branches attached to CD ring depends on the size of CD, and 2 and 3 branches were easily attached to a-CD and p-CD, respectively. Panose was also attached to CDs to form panosyl-CDs. Branching moiety of the CDs were degraded by the action of glucoamylase to glucosyl-CDs (G1-CDs), and maltose was again attached to G1-CDs to form doubly branched G1-, G2-CDs by the action of pullulanase. Properties of branched CDs : Solubility was higher than that of original CDs, and degree of the solubility was different depending on the variety of CD, the variety of branch and the number of branches. As for the action of starch-degrading enzymes on the branches, it was found that the action of glucoamylase was considerably different depending on the variety of branched CD. Amylo-1, 6-glucosidase acted on AD and AC type of diglucosyl-α-CD((G1)2-α-CD), but not on AB type. Aspergillus oryzae α-amylase degraded G1-CD, but the rate of degradation was depressed to less than 1/10 in comparison with that of original CD, and multiply branched glucosyl-CDs were scarcely degradable. Pullulanase (branching enzyme) scarcely degrades maltosyl branches of AB type of branched CDs, but acted on the branches of AC and AD types. AD type of (G1)2-α-CD was completely resistant to the action of macerans enzyme ; AB and AC types were reacted in the presence of acceptors such as glucose and maltose. By the combination of the results described above, systems for the separation and produc-tion of AB, AC and AD types of (G1)2-α-CD were established.

PURIFICATION AND SOME PHYSICAL AND CHEMICAL PROPERTIES OF RED KIDNEY BEAN (PHASEOLUS VULGARIS) ?-AMYLASE INHIBITOR

Red kidney bean contains more amylase inhibitor than do California white bean and cowpea while garbanzo bean and Westan and Westley lima beans do not contain inhibitor. Red kidney bean amylase inhibitor was purified to homogeneity by selective heat treatment (60°C) of a water extract at pH 4. 0, fractionation with ethanol and successive chromatography on DEAE- and CM-cellulose chromatography. The inhibitor has an apparent molecular weight of 49,000 by Sephadex gel filtration and contains 8. 6% carbohydrate probably covalently linked via an amide linkage to asparagine. The inhibitor probably contains four subunits perhaps of three different types. The inhibitor is high in aspartic acid, glutamic acid, serine, threonine and valine, low in cysteine/cystine and does not contain proline. Stable 1:1 complex formation between inhibitor and porcine pancreatic α-amylase was demonstrated by gel filtration on Sephadex G-100. The inhibitor has activity against porcine pancreatic α-amylase, human salivary α-amylase, and Tenebrio molitor (yellow corn meal worm) larval midgut α-amylase but is inactive against Bacillus subtilis α-amylase, Aspergillus oryzae α-amylase, barley α-amylase and red kidney bean α-amylase.

Multimolecular substrate reactions catalyzed by carbohydrases. Aspergillus oryzae α-amylase degradation of maltooligosaccharides

Aspergillus oryzae alpha-amylase degrades maltooligosaccharides by other pathways besides simple glycosidic bond scission. The utilization of the alternate pathways increases with the concentration of substrate implicating a multimolecular substrate mechanism. Reducing-end labeled and uniformly labeled maltooligosaccharides were used to elucidate these alternate degradation mechanisms. Condensation followed by hydrolysis is not a significant pathway. Transglycosylation is concluded to occur, but no single transglycosylation mechanism can account for all of the experimental data for maltotriose degradation. Rather, a combination of transglycosylations must be invoked.

Cyclodextrin Forming Enzyme of Bacillus macerans

A new enzymatic method to determine cyclodextrins was described in this paper. The principle of the method consists of the combination of specific enzyme actions and the determination of reducing value. Cyclodextrins do not show any reducing value, because of no reducing end. In a mixture containing maltosaccharides, a and ?A-cyclodextrin, glucoamylase hydrolyses only maltosaccharides. Reducing value was determined by Somogyi-Nelson method after degradation of maltosaccharides to glucose (GAV). Total cyclodextrin content was obtained by subtraction GAV from total sugar (PS) which was determined by the phenol-H2SO4 method. Aspergillus oryzae α-amylase hydrolysed fast β-cyclodextrin and extremely slow α-cyclodextrin. β-Cyclodextrin and maltosaccharides were degraded to glucose by the combination of glucoamylase and Aspergillus oryzae a-amylase (reducing value of this enzyme system: GTV). Therefore, a-cyclodextrin content was obtained by subtracting GTV from PS, correction of a-cyclodextrin degradation should be done if necessary. Cyclodextrins were determined by this enzymatic method within the relative error of 5% in the mixture of cyclodextrins and oligosaccharides.

Reaction of protein disulfide groups with Ellman's reagent: A case study of the number of sulfhydryl and disulfide groups in Aspergillus oryzae α-amylase, papain, and lysozyme

Ellman's reagent, 5,5′-dithiobis[2-nitrobenzoic acid] (DTNB and abbreviated here as Ø-S-S-Ø), is a standard reagent for the determination of reactive sulfhydryl groups by absorbance measurement at 412 nm. The color produced is due to the formation of a thio anion, 3-carboxylato-4-nitro-thiophenolate (Ø-S −). In the determination of the number of sulfhydryl groups of porcine pancreatic α-amylase, we observed that the protein contained more Ø-S-S-groups than could be accounted for from the original sulfhydryl analysis (reaction 1). We consequently postulated that disulfide groups were reacting in addition to sulfhydryl groups (reactions 2 and 3). The reaction was postulated to release one Ø-S − for every free sulfhydryl group, and the protein was postulated to be derivatized so that one -S-S-Ø group is formed for every free sulfhydryl group and two -S-S-Ø groups are formed for every disulfide group. This hypothesis was tested by using three diverse proteins ( A. oryzae α-amylase, papain, and lysozyme) whose numbers of sulfhydryl and disulfide groups are known. Ø-S − was released from the derivatized protein by incubation at pH 10.5 (reaction 4). Determination of the amount of Ø-S −/mole of protein before and after pH 10.5 permitted determination of the number of sulfhydryl and disulfide groups in the protein. The results of the study of the three proteins confirmed the hypothesis and established methods for the fast and accurate determination of the number of sulfhydryl and disulfide groups in proteins. 1. (1) Protein-S − + Ø-S-S-Ø → Protein-S-S-Ø + Ø-S − 2. (2) Protein-S-S-Protein + Ø-S − → Protein-S-S-Ø + Protein-S − 3. (3) Protein-S − + Ø-S-S-Ø → Protein-S-S-Ø + Ø-S − 4. (4) 3 Protein-S-S-Ø + DTT or pH 10.5 → 3 Protein-S − + 3 Ø-S −