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This study was undertaken to distinguish between normal and diabetic subjects by lectin-glycated hemoglobin interaction. The quantitative precipitin method was performed for the interaction between glucose-specific lectin Concanavalin A (Con A) and the glucose-containing RBC-lysate for the estimation of calculated HbA1c% from a standard curve. The standard curve was prepared by plotting the optical density of the precipitin for the interaction of standard HbA1c concentration with Con A against HbA1c reference standard. The absorbance range of the precipitate was 0.14-0.20 in normal subjects and the corresponding calculated HbA1c% along with plasma glucose (mg/dl) levels was 4.1-5.8% and 82-101 mg/dl respectively. Higher absorbance values, 0.22-0.42, were obtained in diabetic patients when the calculated HbA1c% was 6.3-12.2% and plasma glucose level was 120-292 mg/dl. Almost similar results were observed for HbA1c% (6.1-11.9%) of the same diabetic samples measured by conventional ion-exchange high performance liquid chromatography (HPLC). Excellent correlation coefficients of the two methods from regression analysis graph for normal (r = 0.98) and diabetic patients (r = 0.99) were observed. Furthermore, nondiabetic and diabetic hemoglobin variant subjects showed similar HbA1c% by our lectin-based assay when compared with standard HPLC method. We conclude that this lectin-based assay may be adopted to estimate glycated hemoglobin level in differentiating between normal and diabetic patients. This assay offers a good correlation with standard HPLC method. Moreover; the method is convenient, cheap, and needs no sophisticated instruments.

The present investigation was designed to distinguish prostate cancer and benign prostate hyperplasia by lectin-prostate specific antigen (PSA) binding.The quantitative precipitin method of concanavalin A (Con A)-carbohydrate interaction was explored with the serum PSA of patients suffering from prostatic complications.The carbohydrate content in the precipitate after binding of Con A with serum PSA of prostate cancer was significantly lower than that of benign prostate hyperplasia. This may be due to altered sugar chain structure or less glycosylation of PSA in prostate cancer.We conclude that a serum value3.0 microg/ml of the carbohydrate content of Con A-PSA precipitate indicates strong suspicion for prostate cancer and this cut off level is effective in reducing the rate of unnecessary biopsies in men with total PSA value between 4.0 to 10.0 ng/ml.

A method has been developed to separate and identify thyroglobulin autoantibody (TgAb) and thyroid peroxidase autoantibody (TPOAb) in serum obtained from normal and autoimmune thyroid diseases using phenyl Sepharose CL-4B hydrophobic column. The protein peaks obtained from hydrophobic column were identified as TgAb and TPOAb by comparing the elution profile of commercially purified standard thyroid autoantibodies. The similarity of the inhibitory effects of eluted proteins and of standard thyroid autoantibodies on lectin concanavalin A-RBC interaction confirmed the separation of TPO-Ab and TgAb by the hydrophobic, column. The eluted fractions from the hydrophobic column were estimated by the radio immunoassay (RIA) to confirm the presence of both auto-antibodies. This hydrophobic column method offers an advantage of visual inspection of this autoantibodies by graphic representation of peak height along with their estimation in autoimmune thyroid disorders.

Antibodies against cyclic citrullinated peptide (anti-CCP) were thought to be more specific than rheumatoid factor (RF) for the diagnosis of rheumatoid arthritis (RA). The determination of anti-CCP in addition to RF could be helpful in the serological diagnosis and monitoring of patients with RA. Citrullination of proteins involves the enzymatic conversion of protein containing arginine residues to citrulline residues by the enzyme peptidylarginine deiminase (PAD). The present investigation was undertaken to estimate serum PAD enzyme activity in RA patients with a view to find its importance as a new diagnosis marker in a rheumatology clinic.The activity of the PAD enzyme was measured by spectrophotometric method at 530 nm in sera of control subjects and in patients of RA (Group I: RF negative and CCP positive: Group II: both RF and CCP positive) in terms of citrulline formation using benzoyl-arginine ethyl ester (BAEE) as substrate. Anti-CCP and RF were also estimated in two groups by enzyme immunoassay and immunoturbidimetry for comparison. Clinical variables (duration of morning stiffness, swollen and tender joint counts, patient's assessment of pain) and C-reactive protein were also evaluated.A marked increase in PAD enzyme activity (p0.001) was noted in RA patients in comparison to controls and the level diminished appreciably along with two known serological markers (anti-CCP and RF) after six months of disease modifying antirheumatic drug (DMARD) treatment. The Group II RA patients showed much higher enzyme activity than Group I RA patients. However, clinical variables did not differ significantly between the two Groups of RA patients.We conclude that determination of PAD enzyme activity may be used as an additional marker for monitoring disease progression and regression along with anti-CCP and RF in patients with RA. Moreover, this method is rapid, sensitive, and inexpensive and can be adopted in a laboratory having modest facilities.

This study was undertaken to distinguish between normal and diabetic subjects by lectin–glycated hemoglobin interaction. The quantitative precipitin method was performed for the interaction between glucose‐specific lectin Concanavalin A (Con A) and the glucose‐containing RBC‐lysate for the estimation of calculated HbA1c% from a standard curve. The standard curve was prepared by plotting the optical density of the precipitin for the interaction of standard HbA1c concentration with Con A against HbA1c reference standard. The absorbance range of the precipitate was 0.14–0.20 in normal subjects and the corresponding calculated HbA1c% along with plasma glucose (mg/dl) levels was 4.1–5.8% and 82–101 mg/dl respectively. Higher absorbance values, 0.22–0.42, were obtained in diabetic patients when the calculated HbA1c% was 6.3–12.2% and plasma glucose level was 120–292 mg/dl. Almost similar results were observed for HbA1c% (6.1–11.9%) of the same diabetic samples measured by conventional ion‐exchange high performance liquid chromatography (HPLC). Excellent correlation coefficients of the two methods from regression analysis graph for normal (r = 0.98) and diabetic patients (r = 0.99) were observed. Furthermore, nondiabetic and diabetic hemoglobin variant subjects showed similar HbA1c% by our lectin‐based assay when compared with standard HPLC method. We conclude that this lectin‐based assay may be adopted to estimate glycated hemoglobin level in differentiating between normal and diabetic patients. This assay offers a good correlation with standard HPLC method. Moreover; the method is convenient, cheap, and needs no sophisticated instruments.

A protease inhibitor has been purified by ultracentrifugation, affinity chromatography on trypsin-sepharose 4B, and chromatofocusing on PBE-94 from hemolymph of the scorpion Heterometrus bengalensis. Homogeneity of the protease inhibitor was demonstrated by high performance liquid chromatography (HPLC). The protease inhibitor is a monomeric glycoprotein with a molecular weight of 120,000 dalton, which is stable between pH 4 and pH 8. The molecule inhibits serine proteases like trypsin and alpha-chymotrypsin and shows a noncompetitive mode of inhibition towards trypsin, with a Ki value of 6.1 x 10(-6) mM. Amino acid analysis shows a preponderance of aspartic acid, glutamic acid, serine, and glycine. The protease inhibitor is efficient in inhibiting phenoloxidase activity in both the hemolymph and the isolated phenoloxidase. Melanin synthesis by phenoloxidase may be influenced by this protease inhibitor.

Background : A sugar-binding protein, lectin, was identified in the cystic fluid of brain tumor (glioma). Methods : The protein was purified by high performance liquid chromatography. Results : The molecular weight of the lectin was 29 kDa. The activity of the purified protein was inhibited strongly by p -nitro-β- d -galactose and asialofetuin. The lectin activity was not Ca 2+ - or Mg 2+ -dependent. The antiserum of this pure lectin specifically cross-reacted in the cerebrospinal fluid (CSF) of glioma tumor patients. The same antibody had no reaction with CSF of other neurological patients, normal rat brain tissue extract, oncofetal antigens such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), plant lectins, and other body fluids like plural and ascites. Conclusions : This antilectin antibody may be useful in the evaluation of patients with primary brain tumor (glioma).

The erythroagglutinating activity of purified Vicia faba lectin was enhanced in the presence of gums; gum guar caused the highest enhancement. Circular dichroism probe demonstrated 40-57% beta-conformation and 4-23% alpha-conformation of the lectin at pH 7.2 depending upon the analytical methods used. The beta-conformations of untreated and modified V. faba lectins were increased in the presence of gums. The mixing of gum guar with lectin and with modified lectin, respectively, led to the highest values of beta-conformational change in the protein molecule, thereby increasing the number of receptor sites of the lectin molecule. The enhancement of the activity of V. faba lectin in the presence of gum guar might be due to the conformational change of the protein molecule.

A lectin from early long pod var. of Vicia faba seed has been purified to homogeneity on chitin. The purified lectin is shown to be homogeneous in nature by Bio Gel P - 150 gel filtration, fast protein liquid chromatography and polyacrylamide gel electrophoresis. The lectin is a glycoprotein with molecular weight of 51,000. The lectin molecule is possibly composed of two types of subunits devoid of any covalent linking through sulfhydryl groups, with molecular weights 9,000 and 15,000 respectively in the ratio 2:2. The purified lectin shows a high affinity for N-acetyl-D-glucosamine (GlcNAc). Amino acid analyses show that cysteine and methionine are absent, and a high proportion of aspartic acid and glutamic acid are present in the protein molecule. The extinction coefficient of the purified lectin is 7.22. The lectin behaves as a 'cold agglutinin' displaying stronger agglutination than the naturally occurring ABO agglutinin in the cold.

On binding toVicia faba lectin, the fluorescence of 4-methylumbelliferyl-α-D-glucoPyranoside was quantitatively quenched showing that the interaction of 4-methylumbelliferyl-α-D-glucoPyranoside took Place in a binding environment. The binding of the fluorescent sugar was saccharide sPecific as evidenced by the reversal of 4-methylumbelliferyl-α-D-glucoPyranoside fluorescence quenching by D-fructose. The association constant,K a, values for the 4-methylumbelliferyl-α-D-glucoPyranoside was determined by comPetition study emPloying reversal of fluorescence quenching of 4-methylumbelliferyl-α-D-glucoPyranoside by D-fructose. TheK a value obtained for D-fructose was 1.07 ±0.03 X 104 M-1 and for 4-methylumbelliferyl-α-D-glucoPyranoside was 1.60 ±0.05 X 104 M-1 at 15°C. TheK a values of 2.51 ±0.06 X 104M-1, l.26 ±0.02 X 104 M-1 and 0.56 ±0.01 X 104M-1, resPectively at 10°, 20° and 30°C were obtained from the ChiPman equation. The relative fluorescence quenching, ΔF a, at infinite concentration of the free saccharide sites ofVicia faba lectin [P′] was 93.5% at 30°C and the binding constant for 4-methylumbelliferyl-α-D-glucoPyranoside lectin interaction as derived by Yank and Hanaguchi equation was 0.63 ±0.01 X 104M-1.

Vicia faba lectin contained 40-57% beta-conformation, 4-23% alpha-conformation along with random coil at pH 7.2 depending upon the analytical methods used. The percentage of beta-conformation increased with the addition of N-acetyl-D-glucosamine or methyl alpha-D-mannopyranoside. The structural transitions of V. faba lectin were affected by alkali at pH 9.6 and 10.6. Binding constants and free energy changes for the interaction between V. faba lectin and N-acetyl-D-glucosamine and methyl alpha-D-mannopyranoside were estimated at pH 7.2 using the c.d. and fluorescence methods.

Human erythrocyte specific lectin was isolated from the seeds ofErythrina variegata Linn. var.orientalis Linn. Merrill. The lectin preferentially agglutinated erythrocytes in the sequence of O>B>A = AB. The lectin was purified 19-fold by affinity chromatography on acid treated sepharose 4B with an yield of 81%. The purified lectin was found homogeneous on polyacrylamide gel electrophoresis. The erythroagglutination reaction was inhibited by N-acetyl-D-galactosamine, D-galactose and lactose at very low concentration. The haemagglutination by the purified lectin was not inhibited by different hexose and pentose sugars even at high concentration. The purified lectin was a glycoprotein and agglutinated leucocytes at 3 μg protein concentration. The lectin induced transformation of peripheral blood lymphocytes in cultures.

Two lectins, an N-acetylgalactosamine-binding lectin, lectin-I, which reacts specifically with human erythrocytes of blood group A, and a galactose-binding lectin, lectin-II, which is specific for human blood group B erythrocytes, have been isolated and purified from rice bean, Phaseolus calcaratus syn. Vigna umbellata , by a salt solubility pH-dependent method, chromatofocusing and high-performance liquid chromatography. The homogeneity of the lectins was determined by liquid chromatography and polyacrylamide gel electrophoresis. The purified lectin-I of molecular mass 80 000 is possibly composed of two subunits of molecular mass ca. 18 000 and 22 000, respectively, whereas lectin-II of molecular mass 100 000 appears to be composed of a monomeric protein of molecular mass 25 000. One endogenous lectin-binding protein was also isolated and purified by liquid chromatography. The endogenous lectin-binding protein of molecular mass 40 000 affects the activity of the A-group specific lectin more than that of the B-group specific lectin. The endogenous lectin-binding protein appears to be composed of a monomeric protein of molecular mass 20 000.

An erythroagglutinin from the hemolymph of the scorpion, Heterometrus bengalensis, has been purified by gel filtration and ion-exchange chromatography. Its homogeneity has been demonstrated by polyacrylamide gel electrophoresis. The purified agglutinin appears to be a monomeric protein having a possible molecular weight between 146,000 and 148,000. It has no divalent cation requirement for erythroagglutination. The erythroagglutination is not inhibited by saccharides, glycoproteins and mucin. Identical erythroagglutination pattern is obtained with normal as well as neuraminidase treated erythrocytes.

Acetylcholinesterase (AchE: EC 3.1.1.7) was identified and purified from the hemolymph of the scorpion Heterometrus bengalensis. The purity of the enzyme was determined by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme, determined by sodium dodecyl sulfate-PAGE, was 80,000. The purified AchE hydrolysed acetylthiocholine iodide, but it did not react with butyrylthiocholine iodide. BW284C51, a specific inhibitor of AchE, strongly inhibited the enzyme. The known inhibitor (tetramonoisopropylpyrophosphortetramide) of pseudocholinesterase did not produce any inhibition of the enzyme activity. The purified AchE of scorpion hemolymph was vulnerable to high substrate concentration. The presence of Cu2+ and Ni2+ reduced the enzyme activity, whereas the metal ion, Sn2+, enhanced AchE activity. Ca2+ produced neither inhibition nor activation. (Na+, K+)-ATPase and Mg2+-ATPase activities were greatly enhanced by the purified AchE.