Chymopapain

As well as all the other enzymes in the PLCPs group, chymopapain is a cysteine protease. Proteases are enzymes that hydrolyse peptide bonds between the residues that conform a protein. In every hydrolysis a water molecule is released. Specifically, a cysteine protease is an enzyme which breaks the peptide bond by using the thiol group of a cysteine residue as the nucleophile. In order to hydrolyse, the whole catalytic triad of the enzyme must be used.[15] This is constituted by a cysteine, the Cys159 residue, a histidine, the His203 residue, and a third residue, which tends to be an asparagine, specifically the Asn313 residue.[16] The functional groups used in the reaction are the thiol group of the cysteine and the imidazolium ring of a histidine. The asparagine residue works orientating the imidazolium ring of the histidine.[17]

Chymopapain hydrolysis mechanism step by step.

The mechanism followed is exposed below:[11][18]

1. The thiol group from the cysteine loses a proton, so it becomes negative charged and the amino group of the histidine catches a proton, which gives it a positive charge.
2. The cysteine makes a bond with the carbon breaking the carbon's double bond with oxygen and converting it into a simple bond.
3. The amino group is attracted by the positive charge of the histidine and a bond between these two is formed. The peptide bond is now broken and the carbonyl group is remade.
4. The NH₂R group is released from the histidine. The bond between the thiol group from the cysteine and the carbon is broken and a NHR group replaces it.

When this two bonds are broken, the catalytic triad from the chymopapain is available to be used again.

Chymopapain is no longer used as a standard method to treat chronic low back pain because of its potential side effects.[19][20] Therefore, there is no need to synthesize it artificially. In fact, the sale and distribution of this protein was discontinued in the US in 2003.

Despite the huge amount of successful use to treat herniated disk disease, chymopapain use was discontinued not because of hazards or inadequacies but rather because it is no longer available due to a decision by its sole manufacturer to stop production. Wardlaw D. Sciatica caused by disc herniation: Why is Chymopapain Chemonucleolysis denied to our patients? Int J Spine Surg. 2016 Dec 31;10:44. doi: 10.14444/3044. PMID: 28377858; PMCID: PMC5374990.

However, several studies have demonstrated different successful methods to extract and isolate the protease, which vary among authors.[21][22] The most common procedure is the one described by Baines & Brocklehurst in 1979.[23]

In order to obtain the protein, Carica Papaya fruits are used, as chymopapain is found in its latex. The papayas should be just in the previous step before maturation, which implies an average diameter of 6–10 cm.[24]

Chymopapain extraction and isolation

Some longitudinal incisions of 2mm of depth have to be made through the skin to proceed to the extraction of latex, which can be collected in solid form some minutes after the extraction. The proteases present in the latex of the fruit are inactive precursors that are activated once the papaya is wounded.[25] In 0.3 ml of latex there are about 15 mg of chymopapain.[26]

If we want to conserve the proteolytic properties, latex has to be preserved with sodium metabisulfite and stored at a low temperature of about -10 °C.[27] If used immediately after the incisions, a buffer is added to extract the proteins: EDTA, ammonium sulfate or phosphate buffer all with a concentration of 0.5 mM and a pH of 7.

It is also important to block the thiol functions to avoid air oxidation and the loss of proteolytic activity.[25]

To eliminate organic and insoluble molecules, the sample is first filtered and afterwards centrifuged at 11000g for 30min.[26] The pellet is discarded and the supernatant added to 96% alcohol with a ratio of 1:3.[27] Impurities precipitate and can be eliminated by filtration. Afterwards, (NH₄)₂SO₄ fractioning is done by addition of this substance at a concentration of 0.472 mg/ml.[26] Chymopapain precipitates and can be retrieved through another centrifugation, again at 11000g for 30min. The supernatant is discarded and the ion exchange chromatography can be carried out, with a linear gradient of 100mM (Na⁺) and different volumes of elution. Studying A₂₈₀ chymopapain is found in the fraction of 750-1000 ml.[25]

Once chymopapain has been isolated, it can be crystallized through the gradual addition of sodium chloride at pH 2.0, which can take up to 4 days.[28]

Herniated nucleus pulposus of an intervertebral disc.

Chymopapain

Clinical data
Routes of administration	Injection into intervertebral disc
ATC code	M09AB01 (WHO)
Legal status
Legal status	In general: ℞ (Prescription only)
Identifiers
CAS Number	9001-09-6 Y
ChemSpider	none
UNII	1UK146T40N
ChEMBL	ChEMBL1201626 N
NY (what is this?)  (verify)

Chymopapain is one of the substracts used in chemonucleolysis (a type of percutaneous discectomy).[29] This method was a new proposal to treat primary lumbar intervertebral disc disease using a nonsurgical method. As a matter of fact, the treatment consists on an injection of proteolytic enzymes to dissolve the herniated nucleus pulposus of the intervertebral discs. Purified chymopapain is the main component of the injection, composed basically of 20 mg in five millilitres. It is provided in vials containing 10.000 units of the lyophilized agent with 0.37 mg of disodium edetate,[30] 3.5 mg of cysteine hydrochloride monohydrate and 1.0 mg of bisulfide. All of them work as stabilisers and activators. Sodium hydroxide is in charge of adjusting the PH of the solution. Then, the injection is rehydrated with 5 milillitres of steril water.

A surgeon injects the solution directly into the herniated disc on the spine to dissolve part of it and ease the pain. This process is under fluoroscopic control. Chymopapain is responsible for catalysis, both in vivo and in vitro, a rapid reduction in the viscosity and, as a consequence, the weight of the nucleus pulposus. In fact, it is a depolymerization of the chondromuchoprotein[31] and a decrease in the ability of a disk to imbibe fluid. The dose for a single intervertebral disc is 2 to 4 nanokatals, with a maximum dose per patient of 8 nanokatals. Chymopapain injections are normally given under local, rather than general, anaesthesia.

This enzyme has been studied by universities departments around the world.[32][33][34] It was tested as much in animals as in humans and, very rarely, did it cause serious side effects including paralysis of the legs and death.[35] It could also cause anaphylaxis, but it was only seen in 1% of the patients who received the medication.

The sale and distribution of chymopapain was discontinued in the United States on January 27, 2003, after the company producing it decided to stop selling it worldwide.[36][37]

• Papain

• The MEROPS online database for peptidases and their inhibitors: C01.002
• Data sheet for Papain from BIOZYM