Hi there,

Trypsin is not a metalloprotease  and is actually inhibited by calcium and magnesium. The presence of EDTA is required to chelate divalent ions from the cell culture in order to favor trypsin activity to break up ECM and also to break cell interaction mediated by divalents (cadherin). After trypsinization serum is added to inhibit trypsin (serum contains inhibitors and highlevels of divalent ions).

Hi there,

Trypsin is not a metalloprotease  and is actually inhibited by calcium and magnesium. The presence of EDTA is required to chelate divalent ions from the cell culture in order to favor trypsin activity to break up ECM and also to break cell interaction mediated by divalents (cadherin). After trypsinization serum is added to inhibit trypsin (serum contains inhibitors and highlevels of divalent ions).

Hi,

Trypsin does not require metal so EDTA may not block it. Purpose of adding EDTA is inhibit metalloprotease of cells other than trpsin.

Trypsin is a serine protease, not a metalloprotease.

EDTA addition is to prevent the aggregation of trypsin-treated and floated cells via chelating Ca and Mg.

Trypsin action is inhibited by the addition of serum or serum containing medium.

By the way, human serum has Alpha 2 macroglobulin (inhibit all four classes of proteinases by a unique 'trapping' mechanism) at median value of 2.6 μg/mg of serum protein (range 1.8-13.8 μg/mg of serum protein ; n=13) and Alpha-1 Antitrypsin (serine protease inhibitor; irreversibly inhibit trypsin, chymotrypsin and plasminogen activator) at median value of 1.8 μg/mg of serum protein (range 0.5-3.8 μg/mg of serum protein ; n=13).

16 April 2017  Amended on 21 April 2017

I would like to say thank you very much for kind idea to hydrolase problemn to Dr. Charles Herr (Eastern Washington University, Cheney, WA, USA) that “Trypsin makes more sense to treat with EGTA since it is more selective for calcium, and EDTA is useful for reducing the activity of the trypsin”.   Surely, I agree that protease/peptide-hydrolase have metallo-type characteristics.  I also agree that trypsin makes more sense to treat with EGTA since it is more selective for calcium, and commercial company should sell the trypsin-EGTA in order to use in cell culture.

I have long studied about animal amidase (biotinidase and lipoamidase), and these should be made more sense as called multiple hydrolases.   Importand finding has already been reported by German-American Dr. Hans Neurath (the founding chairman of  Department of Biochemistry, University of Washington, Seattle, WA, USA) as "Neurath H., et al. (1948)  The specific esterase activity of trypsin. Journal of Biological Chemistry, 172, pp. 221-239. (please see file; trypsin esterase)"..   

Then, human serum biotinidase should be called as thiol-type biotinidase/lipoamidase/encephalin-exo-aminopeptidase (this enzyme may have zinc as co-factor although not yet has been measured; please see file; Enk SerumBIN).   Lipoamidase should be called as lipoamidase/de-anchoring enzyme/pancreatic cholesterol-esterase/bile salt-stimulated lipase/BSSL (please see files; Hui lipase, Purify brain lipoamidase, and Multiple hydrolase LIP).   Interestingly, Australian Biochemists Drs. D.H. Small and I.W. Chubb (Department of Biochemistry, University of Melbourne, Parkville, Victoria, Australia) have firstly indicated that acetylcholinesterase has trypsin structure and acetylcholinesterase should be renamed as trypsin/acetylcholinesterase (Small, D. H., & Chubb, I. W. (1988) Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with [3H]diisopropyl fluorophosphate. Journal of  Neurochemistry, 51, pp. 69-74).

Then, notorious PCR method can not give the true values at all, since DNA polymerase enzyme, which also may have the multiple functions, has been used in the PCR method.

Therefore, we have previously used the cell scraper (Corning) to prepare samples to compare protein components between fucoidan-treated and non-treated HepG2 cells (please see file; HepG2 fucoidan).

By the way, protease from invaded virus is dramatically reduced by fucoidan treatment, and the results of PDMD method (new proteomics) have been re-investigated.

Normal Hc has no protease inhibitor, and has only two serine proteases; i.e., Chymase/Mast cell protease I at 12.2 μg/mg of cell protein and Vitamin K-dependent protein C at 4.7 μg/mg of cell protein.

Precise observations are described in the followings.

17 April 2017,  Amended on 18 April 2017

Further, I have studied about the protease excretion from the organs into the blood.   Proteases of invaded microves into blood have been also added.

Serum of healthy 8mo girl (without biotin deficient, with gastritis) has protease inhibitor of Alpha2-Macroglobulin at 1.8 μg/mg serum protein and Alpha1-Antitrypsin at 2.8 μg/mg serum protein (total 4.6 μg/mg serum protein) , but has no proteases (but biotinidase (thiol-type biotinidase/lipoamidase/encephalin-exoaminopeptidase) is present at 4.8 μg/mg serum protein).    Her serum has Replicase polyprotein 1ab (Human coronavirus 229E; HCoV 229E; with thiol-type protease) at 17.7 μg/mg serum protein, and total protease in serum becomes to 17.7 μg/mg serum protein.

Serum of the same girl (12mo, with common cold) has protease inhibitor of Alpha2-Macroglobulin at 13.8 μg/mg serum protein and Alpha1-Antitrypsin at 3.8 μg/mg serum protein (total 17.6 μg/mg serum protein), and esterase of Uncharacterized protein C22orf34 at 17.7 μg/mg serum protein (biotinidase is present at 11.2 μg/mg serum protein).   Her serum has Replicase polyprotein 1ab (Avian infectious bronchitis virus; IBV; with papain-like thiol proteinase) at     9.4 μg/mg serum protein, and total protease in serum becomes to 27.1 μg/mg serum protein (2.7%).

Inflammation may elevate the protease-inhibitor level in the blood.

Serum of biotin-deficient 3y4mo girl (with alopecia and GSD-1b, before biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 2.3 μg/mg serum protein and Alpha1-Antitrypsin at 1.6 μg/mg serum protein, and metalloprotease of A disintegrin and metalloproteinase domain 22/Metalloproteinase-disintegrin ADAM 22-3/ADAM-22 at 2.3 μg/mg serum protein (biotinidase is present at 2.8 μg/mg serum protein).   Her serum has Oligopeptidase A (Haemophilus influenzae; metalloprotease)  at 6.1 μg/mg serum protein and Replicase polyprotein 1a (Murine hepatitis virus; MHV；Murine coronavirus; MCoV; with  papain-like thiol proteinase) at 11.0 μg/mg serum protein, and total protease in serum becomes to 21.0 μg/mg serum protein. 

The same girl after biotin therapy and liver transplantation (4y1mo) has protease inhibitor of Alpha2-Macroglobulin at 3.2 μg/mg serum protein and Alpha1-Antitrypsin at 2.5 μg/mg serum protein, and protease of Cathepsin S at 14.0 μg/mg serum protein and Transmembrane protease, serine 9/Serase-3 at 5.5 μg/mg serum protein (biotinidase is present at 2.8 μg/mg serum protein).   Her serum has Genome polyprotein (HCV; with metalloprotease) at 4.0, Spore protease (Bacillus cereus; with metalloprotease) at 3.9, and UPF0173 metal-dependent hydrolase SE_1382 (Staphylococcus epidermidis) at 10.1 μg/mg serum protein, respectively, and total protease in serum becomes to 37.5 μg/mg serum protein. 

Serum of biotin-deficient 4mo boy (with alopecia, before biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 1.8 μg/mg serum protein and Alpha1-Antitrypsin at 1.0 μg/mg serum protein, and protease of Cysteine protease Atg4D at 4.1 μg/mg serum protein (biotinidase is present at 2.8 μg/mg serum protein).   His serum has ATP-dependent Clp protease ATP-binding subunit clpA  (Escherichia coli) at 3.6 μg/mg serum protein, and Replicase polyprotein 1ab (Feline infectious peritonitis virus; a mutant of FCoV; with thiol-protease) at 3.6 μg/mg serum protein, and total protease in serum becomes to 11.3 μg/mg serum protein. 

The same boy (7mo1w, without alopecia, after biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 6.7 μg/mg serum protein and Alpha1-Antitrypsin at 1.8 μg/mg serum protein, and proteases of Calcium-activated chloride channel regulator 4 at 2.8, Coagulation factor VIII/Antihemophilic factor at 4.9, Matrix metalloproteinase-15/Membrane-type matrix metalloproteinase 2 at 1.9, and Synaptojanin-2 at 3.5 μg/mg serum protein, respectively (biotinidase is present at 3.2 μg/mg serum protein).   His serum has Hemolysin, chromosomal (Escherichia coli) at 5.6 μg/mg serum protein, and total protease in serum becomes to 18.7 μg/mg serum protein.

Serum of biotin-deficient 1y girl (with alopecia, before biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 4.2 μg/mg serum protein and Alpha1-Antitrypsin at 2.7 μg/mg serum protein, and proteases of Angiotensin-converting enzyme/ACE at 7.5 μg/mg serum protein and Probable serine protease HTRA4 at 6.9 μg/mg serum protein (biotinidase is present at 2.8 μg/mg serum protein).     Her serum has no infected protease, and total protease in serum becomes to 14.4 μg/mg serum protein. 

The serum of same girl (1y4mo, without alopecia, after 4mo of biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 4.2 μg/mg serum protein and Alpha1-Antitrypsin at 1.3 μg/mg serum protein, and protease of Complement factor B at 8.0 μg/mg serum protein  (biotinidase is present at 2.7 μg/mg serum protein).   Her serum has Aminopeptidase E (Lactobacillus helveticus; thiol-type) at 9.3 μg/mg serum protein and Genome polyprotein (Soybean mosaic virus) at  10.0 μg/mg serum protein, and total protease in serum becomes to 27.3 μg/mg serum protein. 

The serum of same girl (1y11mo, without alopecia but with anorexia, after 11mo of biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 4.0 μg/mg serum protein and Alpha1-Antitrypsin at 1.4 μg/mg serum protein, and protease of Calpain-9 at 1.6 μg/mg serum protein (biotinidase is present at 3.2 μg/mg serum protein).   Her serum has Genome polyprotein  (Human hepatitis A virus ; HAV; ser-type) at 10.7, Genome polyprotein (HCV; ser-type) at 7.6, Immunoglobulin A1 protease (Haemophilus influenzae; ser-type) at 8.4, Lipase 3 (Candida albicans) at 16.5, Non-structural polyprotein (O'nyong-nyong virus; ONNV; thiol protease) at 1.4, Pol polyprotein (AKR (endogenous) murine leukemia virus;  with aspartyl protease) at 3.4, Replicase polyprotein 1a (Human coronavirus OC43; HCoV OC43; Papain-like thiol proteinase) at 10.5, and γ-D-Glutamate-meso-diaminopimelate muropeptidase lytF (Bacillus subtilis) at 6.2 μg/mg serum protein, respectively, and total protease in serum becomes to 66.3 μg/mg serum protein (6.6%).    Her serum also have Serine proteinase inhibitor 2 homolog first part (Vaccinia virus Copenhagen; VACV; serpin family)  at 10.2 μg/mg serum protein.

Serum of biotin-deficient 32y female (with Gait disorder, before biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 2.4 μg/mg serum protein and Alpha1-Antitrypsin at 0.6 μg/mg serum protein, and proteases of Angiotensin-converting enzyme 2/ACE 2 at 6.4, Invadolysin/Leishmanolysin-like peptidase at 7.0, Laeverin/Aminopeptidase Q at 5.7, Lipase member H at 5.8, and Protein SGT1 at 4.2 μg/mg serum protein, respectively (biotinidase is present at 3.0 μg/mg serum protein).   Her serum has Genome polyprotein (HCV; metalloprotease) at 5.3 μg/mg serum protein and Genome polyprotein (Yellow fever virus; YFV; ser-type) at 6.1 μg/mg serum protein, and total protease in serum becomes to 40.5 μg/mg serum protein (4.1%).

Serum of patients sister with light biotin-deficient 22y female (with light Gait disorder, before biotin therapy) has protease inhibitor of Alpha2-Macroglobulin at 1.8 μg/mg serum protein and Alpha1-Antitrypsin at 2.3 μg/mg serum protein, and proteases of Polyserine protease 2/Polyserase-2 at 12.3 μg/mg serum protein and Proto-oncogene TRE-2/Ubiquitin carboxyl-terminal hydrolase 6 at 2.8 μg/mg serum protein (biotinidase is present at 3.7 μg/mg serum protein).     Her serum has no infected protease, and total protease in serum becomes to 15.1 μg/mg serum protein. 

The serum of their healthy mother (52y) has protease inhibitors of Alpha2-Macroglobulin at 2.6 μg/mg serum protein, Alpha1-Antitrypsin at 1.8 μg/mg serum protein and Uromodulin-like 1/Olfactorin/UMODL1 at 2.0 μg/mg serum protein, and proteases of ATP-dependent C1p protease ATP-binding subunit d1pX-like､mitochondrial at 3.0, Cytosolic carboxypeptidase 2 at 3.9, and Lactotransferrin/Lactoferrin at 2.1 μg/mg serum protein, respectively (biotinidase is present at 4.0 μg/mg serum protein).   Her serum has Replicase polyprotein 1a (Human coronavirus 229E; HCoV 229E; thiol-protease) at 10.3 μg/mg serum protein, and total protease in serum becomes to 19.3 μg/mg serum protein. 

Serum of healthy man (33y) has protease inhibitors of Alpha2-Macroglobulin at 1.9 μg/mg serum protein and Alpha1-Antitrypsin at 0.50 μg/mg serum protein, and proteases of Aminoacylproline aminopeptidase/Xaa-Pro aminopeptidase 1 at 3.3, Calpain-11 at 3.5, Group VI phospholipase A2 at 1.2, Nicastrin at 3.6 μg/mg serum protein, respectively (biotinidase is present at 7.7 μg/mg serum protein).   His serum has no infected protease, and total protease in serum becomes to 11.6 μg/mg serum protein.

Therefore, diseases of common cold, gait disorder, and anorexia (with weight loss) have elevated serum proteases as compared to healthy two persons and one gastritis patient without inflammation, and without considering serum biotinidase levels (one-tailed test; n1=3 (patient), n2=3 (healthy); Mann-Whitney's U test; p < 0.05).   It is noteworthy that an inflammation serum-marker of ceruloplasmin fragment (from 214-) has shown the same result; i.e., common cold at 2.0 μg/mg serum protein, gait disorder at 2.8  μg/mg serum protein, and anorexia at 2.1  μg/mg serum protein, and three controls have no ceruloplasmin fragment (from 214-).   Then, the result is as follows for ceruloplasmin fragment (from 214-); i.e., one-tailed test; n1=3 (patient), n2=3 (healthy); Mann-Whitney's U test; p < 0.05 (please see file; The Fascio Effect).   The appearance of ceruloplasmin fragment (from 214-) in serum may be linked to the increased protease level in inflammatory serum.   Therefore, serum protease-level may become an another serum marker for the inflammation.      

19 April 2017  Amended on 23 April 2017

Since invaded microbes are also present in liver tissue, I have re-evaluated the protease content.   Protease, which has peptidase activity, has been considered.

Interestingly, protease inhibitor is not present in developing fetal Hc cells.

It is interesting that high expression of protease mimics the cancer and medical doctor usually makes a wrong diagnosis.

Normal fetal hepatocyte Hc has three kinds of biotinidase; i.e., Serum (liver-type) biotinidase (Our sequence) at 1.8, Milk biotinidase (our unpublished sequence) at 1.6, and Urine (kidney-type) biotinidase (Our sequence) at 1.8 μg/mg tissue protein, respectively (total biotinidase is at 5.2 μg/mg cell protein).

Interestingly, milk biotinidase (mammary gland biotinidase) is only appeared in Hc cells, and other developed liver cells has only expressed liver biotinidase and kidney biotinidase.

Hc cells express cellular protease of Chymase/Mast cell protease I (serine protease) at 12.2 μg/mg cell protein and Protein C (Trypsin-like serine protease) at 4.7 μg/mg cell protein (total endogenous protease is 16.9 μg/mg cell protein).  

Further, Hc has cysteine-type endopeptidases from invaded microbes; i.e., Genome polyprotein (Dengue virus 92; DENV, serine protease) at 6.5, and Genome polyprotein (ECHOvirus 9; Enteric Cytopathic Human Orphan virus) at 14.5, Genome polyprotein (HCV) at 2.2 μg/mg cell protein, respectively (microbe protease is total 23.2 μg/mg cell protein).

It is interesting that normal developing Hc has no metalloprotease, and it only has serine- and thiol-proteases.

Then, total proteases become 45.3 μg/mg cell protein (4.5%).

Hepatoma HepG2 (adult, American Caucasoid; without fucoidan and cancer state) has only Serum (liver-type) biotinidase at 18.2 μg/mg cell protein.

HepG2 cells express cellular protease of ADAM-TS-19 (metalloproteinase) at 5.6, Metallocarboxypeptidase D/Carboxypeptidase D at 2.4, Transmembrane protease serine 5/Spinesin at 1.3, and A disintegrin and metalloproteinase with thrombospondin motifs 13/von Willebrand factor-cleaving protease/ADAM-TS-13 at 9.2 μg/mg cell protein, respectively (total endogenous protease is 18.5 μg/mg cell protein).   Metalloproteinase is present at 17.2 μg/mg cell protein.  

Further, HepG2 has proteases from invaded microbes; i.e., Genome polyprotein (containing serine protease; HCV) at 7.8, Genome polyprotein (HGV; GV virus-C; GBV-C) at 15.4, and Genome polyprotein (Human poliovirus; PV) at 5.1 μg/mg cell protein, respectively (microbe protease is total 28.3 μg/mg cell protein).

Then, total proteases become 65.2 μg/mg cell protein (6.5%).

Protease inhibitors are also present; i.e., Metalloproteinase inhibitor 4/Tissue inhibitor of metalloproteinases 4 at 0.85 μg/mg cell protein and Complement C4-B/C3 and PZP-like alpha-2-macroglobulin domain-containing protein 3 at 12.0 μg/mg cell protein (total protease inhibitor is 12.9 μg/mg cell protein). 

Hepatoma HepG2 (with fucoidan and cured state) has Serum (liver-type) biotinidase at 9.6 μg/mg cell protein and Urine (kidney-type) biotinidase at 4.8 μg/mg cell protein (total biotinidase is at 14.4 μg/mg cell protein).

Healed HepG2 cells express cellular protease of ADAM-TS-4/Aggrecanase-1/A disintegrin and metalloproteinase with thrombospondin motifs 4 (glutamyl endopeptidase) at 1.9, Endothelin-converting enzyme-like 1 (metalloendopeptidase) at 8.5, Gelatinase B/Matrix metalloproteinase-9 at 2.8, Proteasome subunit beta type-1/Macropain subunit C5 (threonine protease) at 0.26, Matrix metalloproteinase-19 (metalloprotease) at 1.3, Neprilysin (thermolysin-like endopeptidase, metalloprotease) at 1.2, Neuroligin-1 (serine-type carboxylic ester hydrolase) at 5.2, and Serine protease TADG-15/Suppressor of tumorigenicity 14 protein at 1.2 μg/mg cell protein, respectively (total endogenous protease is 22.4 μg/mg cell protein).   Metalloproteinase is present at 15.7 μg/mg cell protein.    Gene expression of Metalloproteinases is not so changed by fucoidan.

Further, cured HepG2 has no proteases (activity possessing) from invaded microbes, and complete disappearance via fucoidan therapy has been observed in proteases from invaded virus.

Then, total proteases in healed HepG2 become 45.2 μg/mg cell protein (4.5%).

Protease inhibitors are also present in healed HepG2; i.e.,Serpin-1/Serine proteinase inhibitor 1 (Variola virus) at 0.62, Amyloid beta A4 protein/Alzheimer disease amyloid protein (serine-type endopeptidase inhibitor) at  0.34, and Serine protease inhibitor Kazar-type 4 at 0.40 μg/mg cell protein, respectively (total protease inhibitor is 1.36 μg/mg cell protein).    Fucoidan has reduced protease inhibitors for 9.5-fold.

These results suggest that fucoidan mainly decreases proteases of invaded virus via reducing invaded virus.   This result strongly supports also the recently declared phrase of "Effect of Fucoidan Is Not Due to Gene Expression" (please see again file; HepG2 fucoidan). 

Liver tissues;  

Normal liver tissue (pseudo cancer) has Serum biotinidase (liver-type) at 22.7 μg/mg tissue protein and Urine (kidney-type) biotinidase at 16.0 μg/mg tissue protein (total biotinidase is at 38.7 μg/mg tissue protein).

Normal liver tissue (pseudo cancer) expresses cellular protease of Ubiquitin carboxyl-terminal hydrolase 36/Ubiquitin thioesterase 36 at 7.4, Acylamino-acid-releasing enzyme/APH at 2.0, β-Secretase 1 (Asp protease) at 3.9, and Complement factor I (serine protease) at 2.0 μg/mg cell protein, respectively (total endogenous protease is 15.3 μg/mg tissue protein). 

Further, normal liver tissue (pseudo cancer) has proteases from invaded microbes; i.e., Gag-Pol polyprotein (HIV-1; Asp protease) at 3.9, Genome polyprotein (Dengue virus type-4; serine-protease) at 5.2, Genome polyprotein (HCV) at 4.4, Genome polyprotein (Japanese encephalitis virus; JEV) at 28.0, and RNA-directed RNA polymerase (Papain-like viral protease; thiol protease; Murine hepatitis virus; MCoV) at 3.2 μg/mg tissue protein (microbe protease is total 44.7 μg/mg tissue protein).

Then, total proteases become 98.7 μg/mg tissue protein (9.9%).     Metalloprotease is not present in this normal tissue, and he has survived.

This tissue has protease inhibitor of Alpha-2-Antiplasmin/Alpha-2-PI/A2AP at 1.2, Alpha-2-Macroglobulin/Alpha-2-M/A2M at 0.98, and Vitamin K-dependent protein S at 2.1 μg/mg tissue protein, respectively (total 4.3 μg/mg tissue protein).    

LC liver tissue (with leprosy) has Serum biotinidase at 23.0 μg/mg tissue protein and Urine (kidney-type) biotinidase at 13.0 μg/mg tissue protein (total biotinidase is at 36.0 μg/mg tissue protein).　

LC liver tissue (with leprosy) expresses cellular protease of Aminopeptidase N/GP150/CD13 (metalloprotease) at 4.9, Bone morphogenetic protein 1/BMP-1 (metallopeptidase) at 5.0, Carboxypeptidase B/Pancreas-specific protein (metalloprotease) at 0.66, Coagulation factor V/Activated protein C cofactor (serine-type endopeptidase) at 5.5, Elastase IIIA/Protease E (serine-type endopeptidase) at 0.66, and Elastase IIIB/Chymotrypsin-like elastase family member 3B/Protease E (serine-type endopeptidase) at 0.66 μg/mg tissue protein, respectively (total endogenous protease is 17.4 μg/mg tissue protein). 

Further, LC liver tissue (with leprosy) has proteases from invaded microbes; i.e., Genome polyprotein (Bean yellow mosaic virus; plant virus; serine-protease) at 1.5, Genome polyprotein (HAV) at 14.4, Genome polyprotein (HCV) at 21.9, and RNA replicase polyprotein (Turnip yellow mosaic virus; TYMV; plant virus; thiol protease) at 16.2 μg/mg tissue protein, respectively (microbe protease is total 54.0 μg/mg tissue protein).     Metalloprotease is 10.6 μg/mg tissue protein.

Then, total proteases become 102.4 μg/mg tissue protein (10.2%).

This tissue has protease inhibitor of Cystatin S and/or SN (Cysteine protease inhibitor) at 0.49 μg/mg tissue protein and Metalloproteinase inhibitor 1/TIMP-1/EPA at 1.6 μg/mg tissue protein  (total 2.1 μg/mg tissue protein). 

LC liver tissue (numbered as No.6) has Serum biotinidase at 25.6 μg/mg tissue protein and Urine (kidney-type) biotinidase at 15.2 μg/mg tissue protein (total biotinidase is at 40.8 μg/mg tissue protein).　

LC liver tissue (numbered as No.6) expresses cellular protease of Coagulation factor V (serine-type endopeptidase) at 3.5, Complement C2 (serine-type endopeptidase) at 1.3, Insulin-degrading enzyme/Insulinase/IDE (metalloprotease) at 4.0, and Macropain subunit C9/Proteasome subunit alpha type-4 (threonine protease) at 0.16 μg/mg tissue protein, respectively (total endogenous protease is 9.0 μg/mg tissue protein).

Further, LC liver tissue (numbered as No.6) has proteases from invaded microbes; i.e., Gag polyprotein (Rous sarcoma virus; RSV; Asp protease) at 18.7, Genome polyprotein (Dengue virus; DENV-4; serine protease) at 0.31, Genome polyprotein (HCV) at 7.9, Genome polyprotein (Louping ill virus) at 6.4, Genome polyprotein (Mosquit cell fusing agent; CFA flavivirus; serine protease) at 48.9, Large tegment protein deneddylase (Herpes simplex virus 1 ; HHV-1; thiol protease) at 19.4, Pol polyprotein (SIV; aspartyl protease) at 7.5, RNA-directed RNA polymerase (Murine coronavirus; MCoV; thiol-protease) at 29.1 μg/mg tissue protein, respectively (microbe protease is total 138.2 μg/mg tissue protein).

Then, total proteases become 188.0 μg/mg tissue protein (18.8%).     Metalloprotease is only 4.0 μg/mg tissue protein.

This tissue has protease inhibitor of Heparin cofactor II/HC-II (serine-type endopeptidase inhibitor) at 1.7, Plasma serine protease inhibitor/PAI-3 at 1.6, and Serine proteinase inhibitor 2 (Variola virus)  at 1.7 μg/mg tissue protein  (total 5.0 μg/mg tissue protein).   This patient of LC tissue seems to be not able to inhibit metalloprotease of his Insulin-degrading enzyme/Insulinase/IDE, but Alpha-2-Macroglobulin/Alpha-2-M in his HCC tissue may have worked and he has fortunately survived.  

HCC liver tissue (numbered as No.6) has Serum biotinidase at 25.4 μg/mg tissue protein and Urine (kidney-type) biotinidase at 9.5 μg/mg tissue protein (total biotinidase is at 34.9 μg/mg tissue protein).　

HCC liver tissue (numbered as No.6) expresses cellular protease of 72 KD type IV collagenase/MMP-2 (metalloprotease) at 0.3, Aminopeptidase N/Microsomal aminopeptidase/GP150 (metalloprotease) at 2.6, Coagulation factor V (serine-type endopeptidase) at 5.6, Complement factor D/Adipsin (serine protease) at 0.21, Elastase IIIA/Protease E (serine protease) at 0.18, and Esterase D/S-formylglutathione hydrolase (serine hydrolase) at 0.11 μg/mg tissue protein, respectively (total endogenous protease is 9.0 μg/mg tissue protein).

Further, HCC liver tissue (numbered as No.6) has proteases from invaded microbes; i.e., Genome polyprotein (Dengue virus; DENV-4) at 19.8, Genome polyprotein (HCV; serine protease) at 0.61, Genome polyprotein (Hog cholera virus; Classical swine fever virus; CSFV) at 0.74, Non-structural polyprotein (Venezuelan equine encephalitis virus; thiol protease) at 0.77, Pol polyprotein (SIV; aspartate protease) at 4.1, Protein G1/Metalloendopeptidase G1 (Vaccinia virus) at 6.7, and Structural polyprotein (O'nyong Nyong virus; ONNV; chymotrypsin-like serine proteinase) at 18.8 μg/mg tissue protein, respectively (microbe protease is total 51.5 μg/mg tissue protein).    Metalloprotease is 9.6 μg/mg tissue protein.

Then, total proteases become 95.4 μg/mg tissue protein (9.5%).

This tissue has protease inhibitor of Alpha-2-Macroglobulin/Alpha-2-M at 0.19 μg/mg tissue protein. 

HCC liver tissue (with PBC) has Serum biotinidase at 11.5 μg/mg tissue protein and Urine (kidney-type) biotinidase at 8.3 μg/mg tissue protein (total biotinidase is at 19.8 μg/mg tissue protein).　

HCC liver tissue (with PBC) expresses cellular protease of Acylamino-acid-releasing enzyme/APH (serine protease) at 6.7, Aminopeptidase N/GP150 (metalloprotease) at 5.4, Subtilisin-like protease PACE4 (serine protease) at 14.7 μg/mg tissue protein, respectively (total endogenous protease is 26.8 μg/mg tissue protein).    Metalloprotease is 12.1 μg/mg tissue protein.

Further, HCC liver tissue (with PBC) has proteases from invaded microbes; i.e., Gag-Pol polyprotein (HIV-1) at 8.6, Gag-Pol polyprotein (HIV-2; aspartic-type endopeptidase) at 3.4, Gag-Pol polyprotein (SIV) at 8.6, Genome polyprotein (Foot-and-mouth disease virus; FMDV) at 15.1, Genome polyprotein (HAV; serine protease) at 24.0, Genome polyprotein (HCV) at 35.4, Genome polyprotein (Hog cholera virus; Classical swine fever virus; CSFV) at 43.6, and Non-structural polyprotein (HEV; thiol protease) at 7.5 μg/mg tissue protein, respectively (microbe protease is total 146.2 μg/mg tissue protein).

Then, total proteases become 192.8 μg/mg tissue protein (19.3%).  

She (with PBC) has sadly deceased, and her HCC tissue has no protease inhibitor.   Then, the patient without protease inhibitor and with plenty of proteases (c.a. 20% of tissue proteins) seems to be easy to die, although developing immortal Hc cells also has no protease inhibitor, and Healed cultured Hepatoma HepG2 (who has also died) has been decreased by fucoidan.   The gene expression and the role of protease inhibitor may become different between tissue state and cultured immortal state.   

Furthermore, presence of metalloprotease is important for the patients to die.

24 April 2017

Moreover, it is noteworthy that proteomics with PDMD method of fetal developmental Hc cells has been considerably difficult as compared to immortal cancer HepG2 cells.   Developmental cell Hc has 35.2% of developmental proteins, which  usually have no glyco-chains and are easy to be hydrolyzed by proteases.

Zum Beispiel, 1st Histone H 3.1 at 46.7, 2nd Protein kinase C eta type at 40.2, 3rd Actinin-associated LIM protein/PDZ and LIM domain protein 3/Alpha-actinin-2-associated LIM protein at 28.5, (Protein FAM8A1/Autosomal highly conserved protein at 27.5), 4th Plancental-specific 1-like protein/Placenta-specific protein 1 at 24.4, 5th Protein SCD6 homolog/Protein FAM61A at 20.1, 6th Desmoyokin/Neuroblast differentiation-associated protein AHNAK at 19.9, (Cytochrome c oxidase subunit 2 at 18.6), (Mitochondrial chaperon BCS1 at 16.8), 7th Dyslexia susceptibility 1 candidate gene 1 protein at 16.2, 8th Protein SSX8/Synovial sarcoma, X breakpoint 8, isoform CRA_a and RWD domain-containing protein 2A at 15.8, (Neutrophil NADPH oxidase factor 4/Neutrophil cytosol factor 4 at 14.6), 10th Histone deacetylase 4/HDAC4 at 14.2, 11th Notch homolog 2 N-terminal-like protein at 13.4,  (Protein regulator of cytokinesis 1 at 13.1), (Glutathione S-transferase A4/GST class-alpha member 4 at 12.9), (Brain-specific angiogenesis inhibitor 2 at 12.9), (Chymase/Mast cell protease I2.2), (Protein janus-A homolog/Phosphohistidine phosphatase 1 at 12.1), 12th Uncharacterized protein C1orf106 isoform 1/Uncharacterized protein EN SP00000373828 at 12.1, 13th T-cell-specific kinase/Tyrosine-protein kinase ITK/TSK at 9.7, 14th Jumonji domain-containing protein 2B/Lysine-specific demethylase 4B at 8.9, and 15th Uveal autoantigen with coiled-coil domains and ankyrin repeats at 8.7 μg/mg of cell protein, etc., respectively.

Therefore, trypsin-EDTA method may be dangerous to culture and analyze the developing normal cells, since EDTA may interfere the differentiation of them.  

25 April 2017  Amended on 01 May 2017

Furthermore, since development is still important in the sucklings, I have searched the milk proteins.   It is found that human breast milk contains many developmental proteins (31%), but bovine milk contains only 2%.

Human breast milk contains such developmental proteins as Tripartite motif-containing protein 2 at 55.0, Vacuolar protein sorting-associated protein 13B at 55.0, Tetraspanin-14 at 33.8, Macrophage myristoylated alanine-rich C kinase substrate at 25.8, Plexin-D1 at 13.2, Dardarin at 12.8, and Laminin subunit alpha-5 at 10.1 μg/mg of milk protein, etc., respectively.

Mother seems to give her baby to grow well via her breast milk.   But, to stop milk giving to her baby seems to be dangerous, since such proteins remains in her mammary gland; i.e., Docking protein 1 at 7.7,  Delangin/Nipped-B-like protein at 6.9, Synaptotagmin-1 at 5.8, DNA helicase V/Far upstream element-binding protein 1 at 4.9,  and Antigen MLAA-22 at 3.2 μg/mg of milk protein, etc., respectively.   Therefore, breast carcinoma may be induced by these remained developmental milk proteins. 

Human breast milk has many protease inhibitors to protect developmental protein in the milk; i.e., α-Lactalbumin (chelator of Ca and Zn) at 156.0, Lactoferrin (chelator of Fe) at 83.0, Olfactorin/Uromodulin-like 1 (chelator of Ca) at 4.5, α-Fetoprotein (chelator of Cu and Ni) at 2.9, and Chondroitin sulfate synthase 1 (chelator of Co, Mn, and Cd) at 2.5 μg/mg of milk protein, respectively.

α-Fetoprotein (chelator of Cu and Ni) in fetal serum may be important as a protease inhibitor.

Therefore, it is now understood why components of milks between bovine and human are very different; i.e., α- S2 Casein and β- Lactoglobulin are only present in bovine milk at 50.3 μg/mg of milk protein and at 27.5 μg/mg of milk protein, respectively.   Lactoferrin concentration is very low in bovine milk at 3.0 μg/mg of milk protein, but human breast milk contains it at 83.0 μg/mg of milk protein.

Then, it is also understood that human breast milk contains human breast milk biotinidase at 5.8 μg/mg of milk protein which may be working as a developmental protein for brain, but milk biotinidase is absent in bovine milk.   It seems that human fetal period may be shorter as compared to bovine's fetal period. 

Both human and bovine has same embryo-fetus period of c.a. 10mo (-10 to 0 mo).   Bovine neonate can walk after 1h of postpartum, but human neonate can walk after 1y of postpartum.   Therefore, human age ranging from -10 to 12mo has to be recognized as the developing period.

Thus, I have now understood that biotin-deficiency with alopecia has only been occurred among the baby of the developing period via developmental protein of KAP (keratin-associated protein) (please see file; JMBT Alopecia).

Thus, the hair growth in the developed old persons (old alopecia patients) should be achieved by other proteins than developmental proteins. 

13 May 2017  Amended on 20 May 2017

Furthermore, it is noteworthy that HuH-7 is able to be cultured on usual non-coated plastic plates, but other cells (HuH-6, HepG2, and Hc) are cultured on collagen-coated plates (please see file; HepG2 fucoidan).   Hc can be grown well on both plates (our recent observation).   HuH-7 may have expressed much amount of collagens.   Hc has Collagen alpha-5 (VI) chain at 3.7, and HepG2 (healed by fucoidan) has Collagen alpha-1 (XVIII) chain at 9.5 and Collagen alpha-3 (IX) chain at 0.61 μg/mg of cell protein, respectively.   Interestingly, HepG2 (without fucoidan; state of cancer) has no Collagen, but instead has Epiplakin/450 kDa epidermal antigen at 32.4, Hornerin at 16.1, Transforming growth factor alpha/TGF-alpha/Protransforming growth factor alpha at 0.2, Transforming growth factor beta receptor type 3/TGF-β receptor type III/TGFR-3/Betaglycan at 2.7 μg/mg of cell protein, respectively.  These non-collagen proteins and TGF-related proteins may have helped to attach the cells to non-coated plastic plates.   However, HepG2 cell-attachment is more clear onto the collagen-coated plates, and the experiment for fucoidan effect has been performed by using the costly collagen-coated plates (please see file; HepG2 fucoidan).   Furthermore, HepG2 (healed by fucoidan) has instead expressed many skin-cell growth factors; i.e., Fibroblast growth factor 23/Phosphatonin at 0.32, Fibroblast growth factor 7/Keratinocyte growth factor/Heparin-binding growth factor 7 at 0.24, Melanocyte protein Pmel 17/Melanocyte protein PMEL at 5.0, Melanoma-associated antigen D4/MAGE-D4 antigen at 1.2, and Vacuolar ATP synthase subunit D homolog at 0.39 μg/mg of cell protein, respectively.   Further, fucoidan has surely reduced the expression of Protocadherins for c.a. 10-fold; i.e., HepG2 (with fucoidan) has Protocadherin alpha-13 at 0.7, Protocadherin alpha-5 at 0.7, and Protocadherin-17 at 1.3 μg/mg of cell protein, respectively (total 2.7 μg/mg of cell protein), on the other hand HepG2 (without fucoidan) has Protocadherin-16/Fibroblast cadherin-1at 23.8 μg/mg of cell protein and Protocadherin-17 at 0.9 μg/mg of cell protein (total 24.7 μg/mg of cell protein).

By the way, we have succeeded in the hair growth in alopecia children with biotin-deficiency (n=3) by the oral free-biotin administration for three months (10 mg/day) (please see file; JMBT alopecia).  

The mechanism by biotin is as follows i.e., increased free biotin in the body cures the membrane protein metabolism of KAP (keratin-associated protein) and the normal development of hair growth is achieved.   However, the recovery of one biotin-deficient patient with alopecia (1y, girl) has shown incomplete hair growth; i.e., her serum at 16w (4mo) of biotin therapy only still has excreted Collagen alpha-1(XVII) chain/120 kDa Linear Ig A disease antigen at 12.8 μg/mg of serum protein, which suggests that her hair-growth is not so perfect as compared to other two alopecial children, and her regular-doctor must have to continue the administration of free-biotin until symptom of excess-biotin have occurred at 11mo of therapy (our unpublished observation).   Her serum at 16w of biotin therapy has Probable protein-export membrane protein secG (Mycobacterium tuberculosis) and Potassium-transporting ATPase B chain (Mycobacterium bovis), which may have linked her excretion symptom of Collagen alpha-1(XVII) chain/120 kDa Linear Ig A disease antigen into blood.   Her serum at 44w (11mo) of biotin therapy still has Pro-epidermal growth factor/Urogastrone at 1.2 μg/mg of serum protein and Latent-transforming growth factor beta-binding protein 2 at 9.6 μg/mg of serum protein, and this excretion or leakage still continued her symptom via Acetolactate synthase (Mycobacterium tuberculosis) and NADH dehydrogenase I subunit H (Mycobacterium sp. JLS).   Therefore, her hair problem may have been rescued by administrating the fucoidan, since fucoidan is an effective drug against Actinomycetes.

However, adult biotin-deficiency patient does not show alopecia at all.   This differences in the growth of the hair of head between children and adults seem to be an important issue, and I have researched for the possible therapy of alopecia in old persons again by using new proteomics of PDMD method.      

Firstly, although PDMD method has not indicated the changes in 3-oxo-5-alpha-steroid 4-dehydrogenase in our specimens (containing no skin sample specimens), Finasteride (Propecia®) and Minoxidil (Rogaine®) may be effective by helping hair follicles (adult hair cells), although some side-effect by these drugs will be occurred.   On the other hand, skin-cell-growth factors such as Fibroblast growth factor 23/FGF-23/Phosphatonin, Fibroblast growth factor 7/FGF-7/Keratinocyte growth factor/Heparin-binding growth factor 7, Melanocyte protein Pmel 17/Melanocyte protein PMEL, and Melanoma-associated antigen D4/MAGE-D4 antigen can be upregulated by fucoidan (derived from Japanese Mozuku (Silky-Mozuku; Nemacystis decipiens) and from Kombu (sea tangle of Laminaria japonica)), which may support the survival of skin cells (please see file; HepG2 fucoidan).

Collagen alpha-1(XVII) chain/120 kDa Linear Ig A disease antigen (present in serum of partially-healed alopecia-patient by free biotin; 1y4mo girl), Collagen alpha-1(XVIII) chain and Collagen alpha-3 (IX) chain (present in healed HepG2 cells by fucoidan), and Collagen alpha-5 (VI) chain/Collagen alpha-1(XXIX) chain/von Willebrand factor A domain-containing protein 4 (present in normal Hc cells) may be important in adult hair growth via help of fucoidan effect.   Hydrophobicity of Collagen alpha-1(XVII) chain is 0.594, that of Collagen alpha-1(XVIII) chain is 0.649, and that of Collagen alpha-5 (VI) chain is 0.532, which is similar to membrane protein KAP (keratin-associated protein) of 0.597 and membrane protein of pig-brain lipoamidase of 0.532.   Biotin is a regulator of biosynthesis of membrane, and may also contribute to hair growth of adult head with the collaboration of fucoidan.

It is noteworthy that collagen-gene expression seems to be ruled by the infected virus; i.e., Collagen alpha-1(XVIII) chain seems to be upregulated by HHV-4, Collagen alpha-3 (IX) chain seems to be upregulated by FluC, Collagen alpha-1(VIII) chain/Endothelial collagen seems to be upregulated by HHV-4, and Collagen alpha-2 (IV) chain seems to be upregulated by FluA.   Therefore, fucoidan may effect on the gene expression of collagen genes via changing the state of symbiotic virus to humans.   I am very grateful to kind suggestions by Dr. Joel Subach (Rutgers, The State University of New Jersey, New Brunswick, NJ, USA) to get these important results.

As others have pointed out, trypsin is a serine endoprotease and doesn't require metal cofactors. EDTA is added in cell suspension to prevent cell-cell and cell-matrix interactions.

Indeed, EDTA can be used for cell suspension without any trypsin, this is useful if you grow the cells to work with proteins of the plasma membrane, that might get digested by trypsin.