T. N. Pattabiraman

Reevaluation of the phenol-sulfuric acid reaction for the estimation of hexoses and pentoses

Evidence is provided to show that in the conventional phenol-sulfuric acid reaction procedure, phenol underwent sulfonation in situ and the phenolsulfonic acid formed decreased the color intensity for hydroxymethyl furfural (HMF), furfural, and many hexoses and pentoses tested. A modified method is described to overcome this problem in which phenol was added after the dehydration of carbohydrates by sulfuric acid and after cooling the system. The color intensity around 475-485 nm for different compounds was fairly proportional to the amount of furfural derivatives (absorption at 310-320 nm) formed from the sugars in the modified method unlike in the conventional procedure. The studies also show that for condensation of HMF derivatives with phenol, heat is not necessary. The color intensity in the modified method also increased compared to that in the conventional method. The increase in the modified method compared to that in the conventional method was 6.0-fold for furfural, 9.1-fold for hydroxymethyl furfural, 3.7-fold for fructose, 2.3-fold for xylose, and 2.0-fold for glucose and arabinose. The possible reasons for this differential increase are discussed.

Further studies on the mechanism of phenol-sulfuric acid reaction with furaldehyde derivatives

Even though the chromogens formed from mannose and galactose showed comparable absorbances at 480 nm in the conventional (developer present during heat of dilution) and modified (developer reacted at room temperature after cooling; epsilon mannose = 13,700, galactose = 14,000) phenol-sulfuric acid reactions, shoulders in the region 420-430 nm were prominent in the former method. Fucose was 10 times less reactive in the modified method (epsilon = 800) than in the conventional method. 2-Formyl-5-furan sulfonic acid reacted equally efficiently in the two methods (epsilon = 40,800). 5-Methyl-2-furaldehyde, unlike the sulfonate derivative or 5-hydroxymethyl-2-furaldehyde, required heat for condensation with phenol. 2-Furaldehyde dimethylhydrazone reacted 25 times better to form a chromogen (epsilon = 40,500) in the modified phenol-sulfuric acid method. The possible roles of intermediates between hexoses and furaldehydes in forming chromogens and the effect of substitution at the 2- and 5-positions of furaldehyde on the rates of condensation with phenol for the observed differences between the conventional and the modified methods are discussed.

A short-duration colorimetric method based on phenol-sulfuric acid reaction for the estimation of glucosylhemoglobin

Hemolysates were treated with HCl (0.18 M)-acetone solution to remove heme and the globin precipitated was washed with acetone. It was dissolved in 1 ml of 0.05 M Tris-HCl, pH 7.0, subjected to heat treatment for 10 min at 100 degrees C to remove traces of acetone, and treated with 0.05 ml of 80% phenol and 3 ml of H2SO4. The color was measured at 480 nm. Glucosylhemoglobin values in control subjects and diabetics were respectively 0.286 +/- 0.051 and 0.513 +/- 0.081 mole hexose/mole hemoglobin. The increase in diabetics was highly significant (P less than 0.001). A good correlation (r = 0.85) between fasting blood sugar values and glucosylhemoglobin level was observed. When globin solution was subjected to 4 hr hydrolysis with HCl-oxalic acid (2 and 1 mole/liter) solution prior to phenol-sulfuric acid reaction, estimated glucosylhemoglobin values increased to 0.720 +/- 0.083 in control subjects and 1.036 +/- 0.115 in diabetics. The possible reasons for this increase are discussed.

Protease inhibitors from jackfruit seed (Artocarpus integrifolia)

Protease inhibitory activity in jackfruit seed (Artocarpus integrifolia) could be separated into 5 fractions by chromatography on DEAE-cellulose at pH 7.6. A minor fraction (I) that did not bind to the matrix, had antitryptic, antichymotryptic and antielastase activity in the ratio 24:1.9:1.0. Fraction II bound least tightly to the ion exchanger eluting with 0.05 M NaCl and could be resolved into an elastase/chymotrypsin inhibitor and a chymotrypsin/trypsin inhibitor by chromatography on either immobilized trypsin or phenyl Sepharose CL-4B. Fractions III and IV eluted successively with 0.10 M NaCl and 0.15 M NaCl from DEAE-cellulose, inhibited elastase, chymotrypsin and trypsin in the ratio 1.0: 0.53:0.55 and 1.0:8.9:9.8 respectively. Fraction V, most strongly bound to the matrix eluting with 0.3 M NaCl and was a trypsin/chymotrypsin inhibitor accounting for 74% of total antitryptic activity. This inhibitor was purified further. The inhibitor with a molecular weight of 26 kd was found to be a glycoprotein. Galactose, glucose, mannose, fucose, xylose, glucosamine and uronic acid were identified as constitutent units of the inhibitor. Dansylation and electrophoresis in the presence of mercaptoethanol indicated that the inhibitor is made up of more than one polypeptide chain. The inhibitor combined with bovine trypsin and bovine α-chymotrypsin in a stoichiometric manner as indicated by gel chromatography. It had very poor action on subtilisin BPN′, porcine elastase, pronase,Streptomyces caespitosus protease andAspergillus oryzae protease. It powerfully inhibited the caseinolytic activities of rabbit and horse pancreatic preparations and was least effective on human and pig pancreatic extracts. Modification of amino groups, guanido groups and sulphydryl groups of the inhibitor resulted in loss of inhibitory activity. Reduction of disulphide bridges, reduction with sodium borohydride and periodate oxidation also decreased the inhibitory activity.

Proteases and protease inhibitory activities in normal mammalian lenses and human cataractous lenses

Trypsin inhibition (reduction in benzoyl arginine p-nitroanilide hydrolysis), elastase inhibition (reduction in succinyl trialanyl p-nitroanilide hydrolysis), and chymotrypsin inhibition (reduction in acetyl tyrosine ethyl ester hydrolysis) by neutral extracts of mammalian lenses were estimated. The activities were found to be markedly elevated in human cortical cataract lenses compared to normal adult lenses (antielastase 7.21 +/- 3.90 units (mean +/- SD) in cataract compared to 1.46 +/- 0.57 in normals; antitryptic, 0.54 +/- 0.38 and 0.12 +/- 0.04; antichymotryptic, 1.03 +/- 0.61 and 0.297 +/- 0.055). Antielastase activity was distinctly higher in adult normal human lenses compared to infant lenses (0.159 +/- 0.068). Elastase- and trypsin-like activities were detected at low levels in all mammalian lenses. Chymotrypsin-like activity could not be observed in the lenses. The cataractous lenses had lower trypsin- and elastase-like activities compared to normal human lenses (elastase 1.20 +/- 0.643 in normal compared to 0.062 +/- 0.035 in cataract; trypsin, 0.367 +/- 0.154 and 0.069 +/- 0.038). The role of protease: inhibitor complexes in the expression of the individual activities and their role in cataractogenesis are discussed.

Purification and properties of an α-amylase inhibitor specific for human pancreatic amylase from proso (Panicium miliaceum) seeds

An α-amylase inhibitor was purified to homogeneity by acid extraction, ammonium sulphate fractionation, chromatography on carboxymethyl-cellulose, diethylaminoethyl-cellulose and Sephadex G-100 from proso grains (Panicium miliaceum). The calculated molecular weight was 14000. The inhibitor was fairly heat stable and stable under acidic and neutral conditions. The factor was more effective by two orders of magnitude in its action on human pancreatic amylase than on human salivary amylase. It did not inhibit onA. oryzae,B. subtilis and porcine pancreatic amylases. Pepsin rapidly inactivated the inhibitor. Chemical modification studies revealed that amino and guanido groups are essential for the action of the inhibitor. The inhibitor was found to protect both human salivary and pancreatic amylases against inactivation by acid. The mode of inhibition was found to be uncompetitive

Proteases in germinating finger millet (Eleusine coracana) seeds

Proteolytic activity was estimated in germinated finger millet seedlings using the endogenous trypsin/amylase inhibitor as substrate and also with haemoglobin and albumin as substrates. The maximal proteolytic activity was observed on the third day of germination. With the inhibitor as substrate, the proteolytic activity was maximal at pH 2.5. The protease that acted on the inhibitor required sulphydryl groups for maximal activity and was suppressed by diazoacetyl norleucine methyl ester and Pepstatin. The protease that acted on haemoglobin with optimum pH of 5.0, was more stable on storage, did not depend on sulphydryl groups for activity and was unaffected by reagents that react with carboxyl groups.

Estimation of serum α2-macroglobulin based on the esterolytic activity of bound α-chymotrypsin

A colorimetric method to estimate α2-macroglobulin (MG) in human serum is described which is based on the capacity of MG: α-chymotrypsin complex to hydrolyze N-acetyl l-tyrosine ethyl ester in the presence of excess crude preparation of a trypsin/chymotrypsin inhibitor from redwood seed. Acetyltyrosine formed was measured using Folins reagent (7). The method was found to be as reliable as, but at least five times more sensitive than the procedures described using trypsin and soybean trypsin inhibitor. MG level is expressed in terms of micrograms of bovine α-chymotrypsin bound. Serum from healthy males had a lower value (150.5 ± 31.9 μg/ml, n = 20) than in females (196.8 ± 40.4, n = 20). No significant difference between the levels in fasting and postprandial conditions was observed.

A colorimetric method for the estimation of serum glycated proteins based on differential reduction of free and bound glucose by sodium borohydride

A new colorimetric method based on the phenol-sulfuric acid reaction is described for the estimation of serum glycated proteins by the differential reduction of free glucose and hexose bound nonenzymatically with 2.0 and 20 mg of NaBH4 in 0.02 ml of serum, respectively, at room temperature for 15 min. The values (microgram hexose/mg protein) in control subjects (n = 60) and diabetics (n = 90) were estimated to be 5.60 +/- 0.85 and 10.8 +/- 1.6, respectively. The increase was highly significant (P less than 0.001) in diabetics. The serum glycated protein levels correlate well with fasting blood sugar values (r = 0.77, P less than 0.001, n = 25). There was also a highly significant correlation between glycated protein level and glycated albumin value in individual serum samples (r = 0.85, P less than 0.001, n = 25). Values of borohydride reducible glyco-groups bound to serum proteins also correlated well with serum glycated protein levels (r = 0.96, p less than 0.001, n = 20) determined by the thiobarbituric acid assay method. The method is found to be simple and rapid, with a coefficient of variations of +/- 3.8%.

Studies on sodium-borohydride-reducible hexose in glucosyl-albumin

Glucosylated albumin of human serum isolated by dye-ligand chromatography on blue Sepharose, was not found to be completely reducible by sodium borohydride. The percentage reducible hexose as judged by phenol-sulphuric acid reaction was in the range of 49.7 +/- 12.8 in control subjects (n = 24) and 53.8 +/- 14.2 in diabetics (n = 50). Increase in the level of total hexose bound to albumin and reducible hexose were equally significant in diabetes (P less than 0.001). Sodium chloride gradient elution during chromatography on blue Sepharose showed that glucosylated albumin had lesser affinity than the native protein to the matrix. It is proposed that an addition product between hexose and albumin is formed during nonenzymatic reaction and this adduct is fairly stable and is not reducible by sodium borohydride.