The Stability and the Stabilization of Testicular Hyaluronidase

Abstract

295including the 300th, produced marked nervous reactions within 24 hours after intracerebral injection. This was true when dilution was carried as high as 1 to 1000. Hamsters exposed by intranasal instillation of 20% brain suspension in amounts as small as 0.06 cc became infected. In exposure by intranasal instillation the incubation period was regularly increased and ranged from 2 to 6 days. Brain suspension from hamsters infected intranasally in the 4th passage titred :0-4-77 by intracerebral injection, whereas the same form of material from the 7th passage titred 103.06 by the same route. These titrations were made with 0.03 cc of the various brain suspension dilutions. Spinal cord of hamsters of the 300th intracerebral passage, injected in 10% suspension intracerebrally, produced typical nervous symptoms. This suspension of virus titred l W 5 , as shown in Table II.Summary. Infected hamster brains from the 300th intracerebral passage and from the 4th intranasal passage titred 10"1.77, whereas infected brains from the 7th intranasal passage titred 10-3.66. Positive Newcastle chicken serum neutralized the virus from the 245th and 300th hamster passage, whereas normal chicken serum had no effect. Nasal instillation trials were conducted from the 16th and 300th hamster passage but were not successful (except for the 1st passage) until after the 245th passage.The incubation period of hamsters infected by intranasal instillation is longer than by intracerebral injection.Hamster-adapted 1\Tewcastle virus was carried through 9 passages intranasally, and brain material from this 9th nasal passage was neutralized by positive Newcastle chicken serum. Hamsters infected in tr anasally and in t r acer ebr all y showed symptoms of irritability followed by involuntary motor reactions and paralysis.Over a period of years, numerous preparations of hyaluronidase have been stored in these Laboratories as bulk powder at room temperature without any appreciable loss in activity. I t has been observed, however, that decreases in *potency occur when the enzyme is stored under other conditions. We have therefore conducted an investigation of the factors which inff uence the skbility of testicular hyaluronidase. This involved the development of an accelerated aging test and the use of stabilizing agents. T' he stability of hyaluronidase in bulk, in aqueous solutiofn and in sterile vials has been studied at lO"G, at room temperature and under conditions of accelerated aging. The influence of various bacteriostatic agents on the lability of the enzyme is reported.Methods. The turbidimetric method of assay employed in this investigation has recently been described in detai1.l This procedure permits the use of hyaluronates of varying degrees of purity as substrates and gives consistent .results because of the stabilization of the protein indicator and the prompt termination of the reaction #by heat. The activity of the enzyme is expressed in turbidity reducing milts (T.R.U.) which are defined as that amount of enzyme which will hydrolyze...

Hyaluronidases

“…The inhomogeneous material could be concentrated another five-fold by electrophoresis but the product was still inhomogeneous in the ultracentrifuge. The stability of partially purified testicular enzyme was investigated by McCullagh et al (74). In view of the instability of the enzyme (page 213), this high activity following very prolonged dialysis against distilled water is surprising.…”

The Effect of Topically Applied Hyaluronidase on Cutaneous Permeability to Certain Substances*

“…In a dry state, it is stable at room temperature for more than one year (1) and if stored in the refrigerator at below 50" F., it is stable indefinitely (2). This results in a disappearance of the characteristic reactions of the intact polymer, the reduction in viscosity being the key to permeability changes.…”