Spectroscopic determination of succinylcholine in dosage forms using eosin Y

Two simple and sensitive analytical assay methods using spectrophotometry and spectrofluorimetry techniques were developed for the estimation of succinylcholine chloride (SUC) in pharmaceutical preparations. The suggested methods are based on the formation of an ion pair complex formed between the drug and eosin Y spectrophotometrically (Method I), or the suppressive effect of succinylcholine on the native fluorescence property of eosin Y (Method II). The spectrophotometric method (Method I) involves measuring the absorbance of the complex between succinylcholine and eosin Y at 550 nm in Britton Robinson buffer of pH 3. However, the spectrofluorimetric method (Method II) involves measuring the quenching effect of the studied drug on the native fluorescence property of eosin Y at the same pH at 550 nm after excitation at 480 nm. The absorbance versus concentration of the drug is rectilinear over the range of 0.5 to 15 μg/ml. The formation constant was 3.5 × 10⁴ and the Gibb's free energy change was ?2.5 × 10⁴ J/mol. In Method II, the relative fluorescence intensity was directly proportional to SUC concentration over the range of 0.05 to 1 μg/ml. The proposed methods allowed a successful application to the estimation of succinylcholine ampoules. An explanation of the reaction pathway was postulated.     

Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

pH-dependence of inhibitory action of eosin Y on ATPase activity of smooth muscle actomyosin complex of the uterus

Research of pH-dependence of inhibitory action of eosin Y (2',4',5',7'-tetrabromofluorescin) on ATPase of contractile proteins of smooth muscles of the uterus has shown that the increase of concentration of this inhibitor (from 0.1 to 10 microM) influenced the profile of pH-dependence of ATPase activity of actomyosin: in the presence of 0.1 microM eosin Y the change of optimal value of pH has been observed in more sour side in relation to the control; at the increase of concentration of eosin Y (from 0.5 to 10 microM) the strongly pronounced optimum of pH is absents in general. The ability of eosin Y to inhibit the ATPase activity of contractile complex is dependent on pH of incubation environment. The change of pH from 6.0 to 7.2 results in a 9-fold decrease of magnitude of apparent constant of inhibition Ki (from 6.5 +/- 0.8 microM to 0.74 +/- 0.07 microM). The obtained results indicate that the diminishing of concentration of H+ in an incubation environment favors the increase of affinity ATPase of actomyosin for eosin Y and prove the important role of ionization processes in the system "enzyme-substrate-inhibitor" for realization of inhibitory action of eosin Y.     

Coomassie blue protein dye-binding assays measure formation of an insoluble protein-dye complex.

The solubility of the protein-Coomassie brilliant blue (CBB) complex formed upon Bradford (Anal. Biochem. 72, 248-254, 1976) or Sedmak and Grossberg (Anal. Biochem. 79, 544-552, 1977) protein assay has been investigated by centrifugation or filtration of the assay mix within 10 min of adding dye reagent. The results show complete loss of color yield in the respective supernates and filtrates. This indicates that the protein-CBB complexes are insoluble at the time of absorbance measurement. Protein solubility in the dye reagent may dictate the relative response of the assay to an individual protein and the requirement for macromolecular structure.     

A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids

We have developed a rapid and sensitive method for detecting ribonuclease (RNAase). The method makes use of a RNa-Pyronine Y complex which has a different absorption spectrium from that of Pyronine Y alone. When the RNA is hydrolyzed by RNAase, the spectrum of the complex changes to that of unbound Pyronine Y. The resultant decrease in absorbance at 572 nm is linear for final RNAase concentrations ranging from 2 to 45 ng/ml. Optimal assay conditions were 11.5 μg/ml Pyronine Y, 0.56 mg/ml RNA, 80 μmol/ml Tris-HCl buffer, pH 7.8 and 2–45 ng/ml RNAase. The effect of complex concentration, PH, molarity and temperature upon the rate of the reaction were determined.The assay is applicable to crude cell-free extracts.     

1,4-Butanediol diglycidyl ether (BDE)-crosslinked PEI-g-imidazole nanoparticles as nucleic acid-carriers in vitro and in vivo

Previously reported 1,4-butanediol diglycidyl ether (BDE) crosslinked PEI (branched polyethylenimine, 25 k) nanoparticles (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) (PN NPs) were reacted with varying proportions of a novel linker, 2-(N-1-tritylimidazol-4-yl)-N-(6-glycidyloxyhexyl)-acetamide (IGA linker, 3), to yield PN-g-imidazolyl nanoparticles (PNIm) with improved transfection efficiency. Here, the IGA linker (3) reacted through an epoxy ring to partially convert the residual 1° and 2° amines present in PN NPs to 2° and 3°, respectively, without altering the total number of amines and additionally incorporating the delocalized positive charge of the imidazolyl moiety. The resulting particles were characterized for their size, zeta potential and DNA complexing ability. PNIm/DNA nanoplexes, in the size range of 120-400 nm, were evaluated for transfection efficiency in HeLa, HEK293 and CHO cell lines, which was found to be ～11, ～2-3 and ～2-17 folds higher than PEI, PN-2 (the best working sample of the PN series) (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) and commercial transfection reagents tested in this study, respectively. Also, flow cytometric analysis showed ～78% (ca.～43% in PN-2) cells transfected with the PNIm 10(6)/DNA complex (the best working sample of the PNIm series) in HEK293 cells. Transfection of GFP specific siRNA in HEK293 cells suppressed the gene expression by ～90% (ca.～70% in PN-2). All the cell lines treated with PNIm/DNA nanoplexes showed >90% viability. In vivo gene expression of luciferase enzyme in Balb/c mice showed highest expression in spleen after seven days.     

An 8- to 10-fold enhancement in sensitivity for quantitation of proteins by modified application of colloidal gold

We have modified the highly sensitive protein assay of C. M. Stoscheck (1987, Anal. Biochem. 160, 301-305), resulting in a further 8- to 10-fold enhancement of sensitivity. This assay, responding to protein quantities with a detection limit of 1 ng, involves the single step of addition of colloidal gold solution, as now commonly used in histochemistry and protein blotting, to the protein sample, followed by simple measurement of the change in absorbance at 590 nm within minutes. By increasing the concentration of the colloidal gold, by using gold sol that has been stabilized with 0.01% polyethylene glycol and adjusted to pH 3.8, and by adapting the assay to microtiter plates, this type of assay can be applied to reliably determine proteins in the complete nanogram range. This assay therefore compares favorably to other assay procedures in terms of rapidity, sensitivity, expense, and lack of interference by many laboratory reagents, although like the others it suffers from the drawback of differences in response of different proteins, which is inherent in dye-binding assays.     

Recombinant thermophilic enzymes for trehalose and trehalosyl dextrins production

Two thermophilic and thermostable enzymes, trehalosyl dextrins forming enzyme (TDFE) and trehalose forming enzyme (TFE), able to convert starch and dextrins to ,-trehalose were recently purified and characterized from Sulfolobales [I. Di Lernia, A. Morana, A. Ottombrino, S. Fusco, M. Rossi, M. De Rosa, Extremophiles, 2 (1998) 409; T. Nakada, S. Ikegami, H. Chaen, M. Kubota, S. Fukuda, T. Sugimoto, M. Kurimoto, Y. Tsujisaka, Biosci., Biotechnol., Biochem., 60 (1996) 267; T. Nakada, S. Ikegami, H. Chaen, M. Kubota, S. Fukuda, T. Sugimoto, M. Kurimoto, Y. Tsujisaka, Biosci., Biotechnol., Biochem., 60 (1996) 263; M. Kato, Y. Miura, M. Kettoku, K. Shindo, A. Iwamatsu, K. Kobayashi, Biosci., Biotechnol., Biochem., 60 (1996) 921; M. Kato, Y. Miura, M. Kettoku, K. Shindo, A. Iwamatsu, K. Kobayashi, Biosci., Biotechnol., Biochem., 60 (1996) 925]. The first enzyme transforms starch and dextrins to the corresponding trehalosyl derivatives, with an intramolecular transglycosylation process, which converts the glucosidic linkage at the reducing end from -1,4 to -1,1. The second, hydrolyzes the -1,4 linkage adjacent to the -1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. Herein, we report the cloning and high level expression of the two enzymes of Sulfolobus solfataricus strain MT4 in Escherichia coli using pTrc expression vector. The yield of TDFE and TFE obtained in this expression system was of 180 U/l and of 3630 U/l of medium, respectively.     

Effect of eosin Y on Ca2+ -independent, Mg2+ -dependent ATPase activity of smooth muscle cell plasma membranes

The effect of eosin Y (2',4',5',7'-tetrabromofluorescin) on basic kinetic parameters of the reaction of Mg2+ -dependent hydrolysis of ATP catalysed "basal" Mg2+ -ATPase myometrial cells plasma membrane has been studied. The eosin Y (10-100 microM) inhibited initial maximal velocity of the "basal" Mg2+ -ATPase of plasma membrane assayed for Mg2+ and ATP. At the same time the given inhibitor reduces the affinity of Mg2+ -ATPase for ATP. However, the difficult effect of the inhibitor action is observed for Mg ions: eosin Y in concentration of 10-50 microM increases the enzyme affinity for the ion-activator, while in concentration of 100 microM the affinity of Mg2+ -ATPase for Mg2+ is reduced. An analysis of eosin Y effect on catalytic efficiency of "basal" Mg2+ -ATPase of plasma membrane has shown, that at saturating concentrations of ATP (1 mM) the enzyme activity is less sensitive to the action of inhibitor. On this basis the conclusion is made that ATP in high concentrations can compete with eosin Y for active centre of Mg2+ -ATPase of smooth muscle cells plasma membrane.     

Circular dichroism spectroscopic studies of native and turnover-modified sulfatase A

In a recent communication, A. Waheed and R. L. Van Etten (1979, Arch. Biochem. Biophys. 195, 248) showed that the sulfatase A of rabbit liver (arylsulfate sulfohydrolase, EC 3.1.6.1), which becomes inactivated as it catalyzes the hydrolysis of substrate, covalently incorporates ³⁵S from nitrocatechol [³⁵S]sulfate during this reaction and at the same time loses most of its secondary structure in solution. Circular dichroism spectra presented here for the native and turnover-modified forms of the sulfatase A of ox liver indicate no difference in the region of the spectrum below 240 nm associated with polypeptide backbone contributions or in the region from 350-250 nm associated with the side-chain chromophore transitions. In addition no differences were evident for the two forms of the ox liver enzyme from ultraviolet absorbance and fluorescence spectroscopy measurements. From these data we conclude that, in contrast to the situation with the rabbit enzyme, there is no loss of secondary structure associated with inactivation of ox liver sulfatase A in the course of enzymic catalysis.     

Estimation of disulfide bonds using 2-nitro-5-thiosulfobenzoic acid: limitations

Recently an elegant method for the quantification of the number of disulfide bonds in proteins and peptides has been reported [T.W. Thannhauser, Y. Konishi, and H.A. Scheraga (1984) Anal. Biochem. 138, 181-188]. The method is based on the quantification of 2-nitro-5-thiobenzoate (NTB) formed from the reaction of 2-nitro-5-thiosulfobenzoate with disulfides in the presence of excess sodium sulfite. Here it is reported that the NTB anion undergoes photochemical reaction with excess sulfite in the system, which results in the rapid disappearance of absorbance at 412 nm in the presence of light. The nonchromophoric derivative of this photochemical reaction is tentatively identified as a sulfo derivative of NTB. Based on these observations it is suggested that, for the quantification of disulfide bonds using the NTSB method, the assay should be carried out in the dark.     

Eosin Y-sensitive ATPase activity in men's spermatozoids as a biochemical test for oligozoospermia

The experiments performed on preparations of spermatozoids of men of reproductive age (27-44-year old) studied the ATPase activity (sensitive to inhibited effects of eosine Y) in both normal and oligozoospermia conditions when treating cell suspension with detergents. The methodical approaches for testing the so-called "common" and eosin Y-sensitive ATP-hydrolase activities in spermatozoids were developed. Saponin, the optimal detergent for permeabilisation of their plasma membrane was chosen using laser-correlational spectroscopia method. Saponin perforates effectively the membranes of spermatozoids, decreasing the average hydrodynamical diameter of cells from 10-15 microm (the spermatozoids themselves) to 3-8 microm (treating cell suspension with 0.05% solution of saponin) and even to 2-3 microm (treating spermatozoids suspension with 0.5% solution of detergent). A non-specific inhibitor of ATP-hydrolase's systems, eosin Y, decreases effectively the ATP-hydrolase activity of intact spermatozoids up to 40%. The exact effect of eosin depends on composition of incubation medium. In the model of extracellular conditions (the optimal concentration of detergent is 0.05%), eosin Y-sensitive ATP-hydrolase's activity of spermatozoids in both normal and oligozoospermia cases is increased by 220-240% (at an average). If enzymatic reaction was performed during intracellular conditions modeling (the optimal concentration of saponin is 0.5%), the increase of eosin Y-sensitive ATPase activity (up to 350-400% in normal conditions, and only to 130-150% in oligozoospermia conditions) was detected. This specificity can be used as easy-to-use clinical test for such pathology of men's reproductive system. Eosin Y inhibited doze-dependently the common ATPase activity in spermatozoids in both normal and with studied pathology. In both cases, after linearization of curves of catalytic titration of ATPase activity with eosin Y in Hill's plot the two-phase dependency, of high and low affinities, was found (the average values of imaginary inhibition constant I(0,5) are 0.1 and 0.3-0.4 mM correspondingly). In both normal and oligozoospermia conditions, the high-affinity component has a positive cooperativity, while the low-affinity component is characterized by a negative cooperativity. The obtained results may be of both theoretical and practical value for further investigation of membrane mechanisms used in the support of ion homeostasis in men's spermatozoids and its violation in conditions under different pathological states. Besides, the results can be used as a theoretical basis for improvement of simple and accessible clinical biochemical methods used for testing such a pathology as oligozoospermia.     

UCHL1 S18Y variant is a risk factor for Parkinson's disease in Japan

ABSTRACT: BACKGROUND: A recent meta-analysis on the UCHL1 S18Y variant and Parkinson's disease (PD) showed a significant inverse association between the Y allele and PD; the individual studies included in that meta-analysis, however, have produced conflicting results. We examined the relationship between UCHL1 S18Y single nucleotide polymorphism (SNP) and sporadic PD in Japan. METHODS: Included were 229 cases within 6 years of onset of PD, defined according to the UK PD Society Brain Bank clinical diagnostic criteria. Controls were 357 inpatients and outpatients without neurodegenerative disease. Adjustment was made for sex, age, region of residence, smoking, and caffeine intake. RESULTS: Compared with subjects with the CC or CA genotype of UCHL1 S18Y SNP, those with the AA genotype had a significantly increased risk of sporadic PD: the adjusted OR was 1.57 (95% CI: 1.06 to 2.31). Compared with subjects with the CC or CA genotype of UCHL1 S18Y and the CC or CT genotype of SNCA SNP rs356220, those with the AA genotype of UCHL1 S18Y and the TT genotype of SNP rs356220 had a significantly increased risk of sporadic PD; the interaction, however, was not significant. Our previous investigation found significant inverse relationships between smoking and caffeine intake and PD in this population. There were no significant interactions between UCHL1 S18Y and smoking or caffeine intake affecting sporadic PD. CONCLUSIONS: This study reveals that the UCHL1 S18Y variant is a risk factor for sporadic PD. We could not find evidence for interactions affecting sporadic PD between UCHL1 S18Y and SNCA SNP rs356220, smoking, or caffeine intake.     

Protective Effects of Asiatic Acid on Rotenone- or H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Injury in SH-SY5Y Cells

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a prevalence of 1–2% in people over the age of 50. Mitochondrial dysfunction occurred in PD patients showing a 15–30% loss of activity in complex I. Asiatic acid (AA), a triterpenoid, is an antioxidant and used for depression treatment, but the effect of AA against PD-like damage has never been reported. In the present study, we investigated the protective effects of AA against H₂O₂ or rotenone-induced cellular injury and mitochondrial dysfunction in SH-SY5Y cells. Mitochondrial membrane potential (MMP) and the expression of voltage-dependent anion channel (VDAC) were detected with or without AA pretreatment following cellular injury to address the possible mechanisms of AA neuroprotection. The results showed that pre-treatment of AA (0.01–100 nM) protected cells against the toxicity induced by rotenone or H₂O₂. In addition, MMP dissipation occurred following the exposure of rotenone, which could be prevented by AA treatment. More interestingly, pre-administration of AA inhibited the elevation of VDAC mRNA and protein levels induced by rotenone(100 nM) or H₂O₂ (300 μM).These data indicate that AA could protect neuronal cells against mitochondrial dysfunctional injury and suggest that AA might be developed as an agent for PD prevention or therapy. Special issue article in honor of Dr. Akitane Mori.     

Microspectrophotometric Studies of Romanowsky Stained Blood Cells. III. The Action of Methylene Blue and Azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

Spectrophotometric Characteristics and Assay of Biological Stains III. The Xanthenes

In this continuation paper of the work on the chemical and spectrophotometic characteristics of commercial stains, data on the xanthene dyes are presented. In the xanthene group of dyes, it has been found possible to assay pyronin B, eosins B and Y and ethyl eosin by spectrophotometric means. Phloxine B, rose Bengal, and erythrosin B are assayed by die color acid precipitation method. Typical absorption curves are given for these dyes as well as representative spectral and assay data.     

Microspectrophotometric studies of Romanowsky stained blood cells. III. The action of methylene blue and azure B

The performances of two standardized Romanowsky stains (azure B/eosin and azure B/methylene blue/eosin) have been compared with each other and with a methylene blue/eosin stain. Visible-light absorbance spectra of various hematological substrates have been measured. These have been analyzed in terms of the quantities of bound azure B, methylene blue and eosin dimers and monomers, and in terms of the CIE color coordinates. It has been found that the addition of methylene blue to azure B/eosin produces little change in performance, at least using these two analytical methods. Methylene blue/eosin does not produce the purplish colorations typical of the Romanowsky effect. This is due not to differences between the spectra of methylene blue and azure B, but to the fact that methylene blue does not facilitate the binding of eosin to cellular substrates to the same extent as azure B.     

Estimation of the membrane permeability of liposomes via use of eosin Y chemiluminescence catalysed by peroxidase encapsulated in liposomes.

The initial rate of horseradish peroxidase (HRP)-catalysed chemiluminescence (CL) reaction in an aqueous compartment of liposomes was applied to the estimation of membrane permeability of liposomes. HRP-encapsulated liposomes were prepared by an extrusion method, and a CL reagent and H(2)O(2) were added into the liposomes suspensions. Fluorescein, eosin Y and phloxin B, which are xanthene dyes with different chemical structures, were used as CL reagents. Xanthene dye and H(2)O(2) permeate into the inner phase of liposomes, resulting in initiation of the HRP-catalysed xanthene dye CL reaction with H(2)O(2). The initial rate of the CL reaction was independent of the xanthene dye used. The reproducibility of the initial rate with eosin Y was better than that with fluorescein and phloxin B. When the membrane permeability of the liposomes was changed by altering the concentration of cholesterol in them, the initial rate of the eosin Y CL reaction was dependent on the membrane permeability of the liposomes.     

Understanding Romanowsky staining. I: The Romanowsky-Giemsa effect in blood smears

Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of 'toxic' granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).     

Determination of reduced disulfide groups in monoclonal antibodies

Reduction of disulfide bonds to sulfhydryl groups for direct radiolabeling of antibodies for immunoscintigraphic and therapeutic applications continues to be of considerable interest. Sensitive spectrophotometric methods have been evaluated that will enable investigators to determine submicrogram quantities of cysteine units produced, for the assurance of controlled reduction. One method, which generates a cysteine-ninhydrin complex (520 nm), has a molar extinction coefficient of 30 250 and can determine 0.04 micrograms/ml cysteine units with an absorbance of 0.01. The method has been applied to determine the quantity of cysteine groups produced by the reduction of an immunoglobin G antibody with five different reducing agents in normal to five times the previously determined optimal molar ratios. The quantities of cysteine units produced from the controlled reduction from 240 micrograms immunoglobin G ranged from 0.073 +/- 0.01 to 1.07 +/- 0.04 micrograms, which were merely 0.54 +/- 0.08% to 7.9 +/- 0.28% of the total available disulfide groups in the protein.