Release of amino acids from soil humic acids by proteolytic enzymes

Soil Research ◽  
1967 ◽  
Vol 5 (2) ◽  
pp. 161 ◽  
Author(s):  
JN Ladd ◽  
PG Brisbane
Keyword(s):  
Amino Acids ◽  
Acid Hydrolysis ◽  
Humic Acids ◽  
Direct Evidence ◽  
Proteolytic Enzymes ◽  
Approximate Inverse ◽  
Peptide Bonds ◽  
Aromatic Content ◽  
Phenolic Polymer ◽  
Soil Humic Acids

Direct evidence for the occurrence of peptide bonds in soil humic acids has been obtained by showing that the proteolytic enzyme pronase releases a-amino acids from each of 10 humic acids. The maximal amounts of amino acids liberated by pronase accounted for 27.6-39.4% of those released by hot acid hydrolysis. The susceptibility of humic acids to proteolytic attack showed an approximate inverse relationship with their aromatic content, based on their absorbance at 260 or 450 m�. Both pronase and papain attacked casein less readily when it was incorporated into a phenolic polymer prepared from p-benzoquinone. Papain was inactive towards soil humic acids. Similar amino acids were released by pronase from each of five humic acids tested. The amino acid patterns showed no basic amino acids but relatively high proportions of leucine (isoleucine) as compared with those obtained by acid hydrolysis. The action of pronase on humic acids had little effect on the molecular weight distribution of the humic components.

Comparison of some properties of soil humic acids and synthetic phenolic polymers incorporating amino derivaties

Soil Research ◽  
1966 ◽  
Vol 4 (1) ◽  
pp. 41 ◽  
Author(s):  
JN Ladd ◽  
JHA Butler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acid Hydrolysis ◽  
Humic Acids ◽  
Gel Filtration ◽  
Quinone Imine ◽  
Peptide Bonds ◽  
Molecular Weights ◽  
Soil Humic Acids ◽  
Properties Of Soil

Twenty-three model phenolic polymers, either nitrogen-free or incorporating amino acids, peptides, or proteins, have been prepared from p-benzoquinone and catechol under mild oxidative conditions. Two lines of experimentation have demonstrated properties of soil humic acids closely similar to those of polymers incorporating proteins, but different from those of polymers incorporating amino acids: (1) fractionation of humic acids and synthetic polymers by 'Sephadex' gel filtration showed that the percentage of components of molecular weights nominally greater than 100 000 ranged from 52-76 % for eight humic acids tested, 53-59 % for benzoquinone-protein polymers (excluding polymers containing protamine), but less than 20% for all other polymers; (2) acid hydrolysis with 6M HCl resulted in a partial release of polymer nitrogen. Amino acid nitrogen in the hydrolysates accounted for 32.4-51.9 % of humic acid nitrogen, 31.2-56.3 % of the nitrogen of polymers incorporating protein, but less than 10.8% of the nitrogen of polymers incorporating individual amino acids. Experiments with model monomeric N-phenylglycine derivatives and with polymers incorporating simple peptides showed that the bond between the carbon atom of an aromatic ring and the nitrogen atom of an a-amino acid is far more stable to acid hydrolysis than peptide bonds or bonds linking amino acids in humic acids. Glycine is, however, readily released from N-phenylglycine derivatives when conditions favour their oxidation to a quinone-imine intermediate. Incorporation of proteins into phenolic polymers prevented the detection of peptide bonds by the Folin reagent.

ACTION OF PROTEOLYTIC ENZYMES ON SOIL ORGANIC MATTER

1970 ◽  
Vol 50 (2) ◽  
pp. 233-241 ◽  
Author(s):  
F. J. SOWDEN
Keyword(s):  
Amino Acids ◽  
Organic Matter ◽  
Amino Acid ◽  
Soil Organic Matter ◽  
Acid Hydrolysis ◽  
Leucine Aminopeptidase ◽  
Proteolytic Enzymes ◽  
Complexing Agent ◽  
Organic Matter Fractions ◽  
Hydrolysis Of

The amino acids set free by proteolytic enzymes were determined with an amino acid analyzer. Soil and enzyme blanks were subtracted. Pronase released 2 to 10% of the aspartic acid + asparagine, threonine, serine, glutamic acid + glutamine, glycine, lysine and histidine in some fractions of soil organic matter along with 15–35% of the alanine, valine, isoleucine, leucine, tyrosine, phenylalanine and arginine. There was no release of proline, ornithine or ammonia. When the pronase hydrolysate was treated with leucine amino-peptidase, 15% of the proline was released, the yield of glycine was raised from 2 to 14% and the amount of the acidic amino acids was also higher. Acid hydrolysis of the pronase hydrolysate also released more amino acid material but the blanks were much higher than with leucine aminopeptidase. The results suggested that more than half of the aspartic and glutamic acids found on acid hydrolysis were present in the soil organic matter fractions as asparagine and glutamine. The action of pronase on the organic matter of the intact soil was slight, even in the presence of a complexing agent. Papain released very little amino acid material from organic matter fractions, but leucine aminopeptidase or HCl hydrolysis of the papain hydrolysate released about 10% of the amino acid of the fraction, indicating that significant amounts of peptides were formed on papain treatment.

scholarly journals The Proteolytic Activity of Diapausing and Newly Hatched Red Mason Bees (Osmia Rufa: Megachilidae)

2014 ◽  
Vol 58 (1) ◽  
pp. 85-91
Author(s):  
Ewa A. Zaobidna ◽  
Kamila Dmochowska ◽  
Regina Frączek ◽  
Ewa Dymczyk ◽  
Krystyna Żółtowska
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Proteolytic Enzymes ◽  
Activity Levels ◽  
Solitary Bee ◽  
Peptide Bonds ◽  
Short Side ◽  
Male And Female ◽  
Substrate Preferences ◽  
Males And Females

Abstract Osmia rufa is a solitary bee that is used commercially for pollinating crops. The bee enters obligatory diapause as an imago. The activity of proteolytic enzymes during diapause has not been investigated. We studied the proteinase activity on four substrates - casein, haemoglobin, bovine serum albumin (BSA ), and gelatine - during diapause (from October to March) and in newly hatched males and females in April. During diapause, greater fluctuations in enzyme activity levels were noted in males than in females, and a significant decrease in male enzyme activity was observed in January and March. Male enzymes were most effective in decomposing gelatine; whereas, female enzymes were equally effective in hydrolysing gelatine and BSA . The differences in substrate preferences between male and female enzymes were particularly pronounced in October and in the newly hatched individuals. The levels of gelatinolytic activity likely indicate that a high proportion of proteinases in O. rufa are elastase-like enzymes. They are involved in the digestion and remodelling of proteins with numerous peptide bonds formed by amino acids with short side-chains.

scholarly journals The amino acid sequence of dromedary pancreatic ribonuclease.

1975 ◽  
Vol 147 (3) ◽  
pp. 505-511 ◽  
Author(s):  
G W Welling ◽  
G Groen ◽  
J J Beintema
Keyword(s):  
Amino Acids ◽  
Polypeptide Chain ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Pancreatic Tissue ◽  
British Library ◽  
Peptide Bonds ◽  
Pancreatic Ribonuclease ◽  
External Loop

Dromedary (Camelus dromedarius) RNAase (ribonuclease) was isolated from pancreatic tissue by affinity chromatography. Peptides obtained by digestion with different proteolytic enzymes and CNBr were isolated by gel filtration, preparative high-voltage paper electrophoresis and paper chromatography. Peptides were sequenced by the dansyl-Edman method. All peptide bonds were overlapped by one or more peptides. The polypeptide chain consists of 123 amino acids. A deletion (position 39) was observed in an external loop of the polypeptide chain (residues 35-40), as was found earlier to horse RNAase (Scheffer & Beintema, 1974). A heterogeneity was found at position 103 (glutamine and lysine). Dromedary RNAase differs at 23-32% of the positions from all other pancreatic RNAases sequenced to date. In evolutionary terms this indicates that dromedary RNAase has evolved independently during the larger part of the evolution of the mammals. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50046 (14 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.

Studies on soil humic acids: I. The chemical nature of humic nitrogen

1955 ◽  
Vol 46 (2) ◽  
pp. 247-256 ◽  
Author(s):  
J. M. Bremner
Keyword(s):  
Amino Acids ◽  
Humic Acid ◽  
Nitrogen Content ◽  
Acid Hydrolysis ◽  
Humic Acids ◽  
Chromatographic Analysis ◽  
Chemical Nature ◽  
Diaminopimelic Acid ◽  
Amino Sugars ◽  
Different Soils

1. The chemical nature of the nitrogen in humic acid preparations isolated from 0·5M-sodium hydroxide and 0·1M-sodium pyrophosphate (pH 7·0) extracts of nine different soils has been studied by determining the amounts of acid-soluble-N, ammonia-N, amino-sugar-N and α-amino-N liberated by acid hydrolysis of the preparations and by paper chromatographic analysis of their acid hydrolysates.2. Humic acid preparations isolated from alkali and pyrophosphate extracts of the same soil differ markedly in total nitrogen content and in nitrogen distribution after acid hydrolysis. The alkali-extracted preparations have a higher nitrogen content and a higher proportion of acid-soluble-N and α-amino-N.3. A considerable fraction (20–60%) of the nitrogen in the preparations examined was not dissolved by acid hydrolysis. The major fraction of the nitrogen dissolved was in the form of amino-acids.4. At least 31–48% of the nitrogen in the alkaliextracted preparations and 20–35% of the nitrogen in the pyrophosphate-extracted preparations was in the form of protein. From 3 to 10% of the nitrogen in the preparations was in the form of amino-sugars.5. The results obtained by paper chromatographic analysis of acid hydrolysates of the preparations indicated that the protein materials in humic acids isolated from different soils by alkali or pyrophosphate are similar in their amino-acid composition. The following nineteen amino-acids were detected in every hydrolysate examined: phenylalanine, leucine, isoleucine, valine, alanine, glycine, threonine, serine, aspartic acid, glutamic acid, lysine, arginine, histidine, proline, hydroxyproline, α-amino-n-butyric acid, β-alanine, γ-aminobutyric acid and tyrosine. Two unidentified ninhydrin-reacting substances, oxidation products of cystine and methionine, and amino-sugars were also detected in every hydrolysate examined. A third unidentified ninhydrin-reacting substance and a substance provisionally identified as α,ε-diaminopimelic acid were found in some of the hydrolysates.

Inhibition and simulation of proteolytic enzyme activities by soil humic acids

Soil Research ◽  
1969 ◽  
Vol 7 (3) ◽  
pp. 253 ◽  
Author(s):  
JN Ladd ◽  
JHA Butler
Keyword(s):  
Humic Acid ◽  
Humic Acids ◽  
Metal Ion ◽  
Proteolytic Enzymes ◽  
Relative Effectiveness ◽  
Trypsin Activity ◽  
Carboxypeptidase A ◽  
Molecular Weights ◽  
Soil Humic Acids ◽  
Stimulation Of

The effects of neutralized solutions of soil humic acids on the activities of a range of proteolytic enzymes and of tyrosinase have been measured. Humic acids inhibit carboxypeptidase A, chymotrypsin A, pronase, and trypsin activities, stimulate papain, ficin, subtilopeptidase A, and thermolysin activities, and had no effect on phaseolain and tyrosinase activities. Stimulation of papain and ficin activities is not due to formation of metal ion-humic acid complexes. Inhibition of trypsin activity and stimulation of papain activity increased with increasing molecular weight of the humic acid. However, humic acid fractions of varying molecular weights had similar effects on the magnitude of inhibition of pronase and carboxypeptidase A activities. Polycondensates derived from p-benzoquinone and catechol influenced enzyme activity in the same way as humic acids, although the order of their relative effectiveness changed with different enzymes. Polyacrylate preparations were the most effective inhibitors of trypsin activity but had no effect on papain and ficin activities.

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