Ultraviolet spectral changes during chymotrypsinogen activation

Chervenka, Charles H.

doi:10.1016/0006-3002(57)90084-7

Cited by 19 publications

(8 citation statements)

References 11 publications

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“…Ovalbumin contains nine tyrosine residues (Tristram, 1953), of which only about two titrate in the pH range of 9.5-12 (Crammer and Neuberger, 1943); there is time-dependent ionization above pH 12.3 (Tramer and Shugar, 1959); and at pH 13, the ionization is instantaneous (Crammer and Neuberger, 1943). The titration curve of ovalbumin in 1 m KHCOs buffer containing 10% dioxane showed no significant differences from that reported by Crammer and Neuberger (1943) in the pH range 8.3-11.8; however, timedependent ionization manifests itself already at pH 11.5. Above pH 11.8,10% dioxane destabilizes the structure to such an extent that the titration curve is displaced by about 1 pH unit.…”

Section: Resultscontrasting

confidence: 51%

“…The ultraviolet spectra were recorded against protein solutions dissolved in 0.1 m phosphate buffer (pH 7.0) in the case of ovalbumin and in 0.001 n HC1 in the case of chymotrypsinogen and trypsinogen immediately after mixing and at 10-min periods thereafter. The optical density within 3 min after mixing was taken as the final value for ovalbumin at pH values above 13 (Crammer and Neuberger, 1943). For chymotrypsinogen and trypsinogen, optical densities after 15 min were taken as the final values at pH 13.5.…”

Section: Methodsmentioning

confidence: 99%

“…The maxima of the difference spectra were found to be at 297 mg for ovalbumin and at 296 mg for chymotrypsinogen and trypsinogen. The number of ionized tyrosine residues was calculated for ovalbumin and trypsinogen using an extinction coefficient of 2300 for the phenoxide ion (Crammer and Neuberger, 1943;Sage and Singer, 1962); for chymotrypsinogen, the corresponding extinction coefficient was taken as 2400 (Marini and Wunsch, 1963).…”

Section: Methodsmentioning

confidence: 99%

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Exposure of tyrosine residues in proteins. III. Reaction of cyanuric fluoride and N-acetylimidazole with ovalbumin, chymotrypsinogen, and trypsinogen

Gorbunoff¹

1969

Biochemistry

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Section: Resultscontrasting

confidence: 51%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Exposure of tyrosine residues in proteins. III. Reaction of cyanuric fluoride and N-acetylimidazole with ovalbumin, chymotrypsinogen, and trypsinogen

Gorbunoff¹

1969

Biochemistry

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“…957) was purchased from Armour Pharmaceutical Co. Ltd., Eastbourne, Sussex. Solutions were prepared in the same way as with chymotrypsinogen, the same extinction coefficient being used (Chervenka, 1957). Trypsinogen.…”

mentioning

confidence: 99%

The reactions of inter- and intra-chain disulphide bonds in proteins with sulphite

Cecil¹,

Wake²

1962

Biochemical Journal

146

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The reaction is strictly reversible and will go to completion only under conditions where the concentration of RS-is small, as for instance at pH 7 (Stricks & Kolthoff, 1951; Cecil & McPhee, 1955). Insulin has two disulphide bonds linking the A and B chains and a third intra-chain bond linking residues 6 and 11 (referred to below as the 6-11 intra-chain bond) on the A chain (Ryle, Sanger, Smith & Kitai, 1955). Cecil & Loening (1960) studied the reaction of these disulphide bonds with sulphite. They found that 'the two inter-chain bonds react with sulphite at pH 7 causing the insulin to split into its component A and B chains. The 6-11 intra-chain bond does not react with sulphite alone but it will react with sulphite in the presence of phenylmercuric hydroxide. Partial reaction occurs in the presence of urea or guanidine. The explanation suggested by Cecil & Loening for this difference in the reactivity of the inter-and intra-chain disulphide bonds was that the structure of the 6-11 intra-chain ring is stabilized by hydrogen bonds (or other non-covalent bonds) rather than by the disulphide bond. This would have the effect of shifting the equilibrium of the reaction with sulphite to the left since the ring would have no tendency to open when the disulphide bond is split. Phenylmercuric hydroxide would block the reverse reaction by combining with the thiol formed. Urea and guanidine would reduce the stability of the 6-11 intra-chain ring by breaking the non-covalent bonds. In these cases the disulphide bond would be expected to react. Bovine serum albumin has 17 disulphide bonds and these appear to behave similarly to the 6-11 intra-chain bond in insulin. Kolthoff, Anastasi & Tan (1958, 1959) found that there was no appreciable reaction with sulphite alone but that all 17 bonds reacted with sulphite in the presence of mercuric chloride. In the presence of guanidine 12-13 bonds reacted with sulphite. Hunter & McDuffie (1959) have shown that the molecular

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“…Most of these researches on various proteins, report peaks at 279 and 287 nm, attributed mainly to the perturbation of tyrosine residues during the unfolding of the polypeptide chain, and at even longer wavelengths, i.e. at around 292-295 nm, when the protein under study contains enough tryptophan residues for an expression of their perturbation as an even more red-shifted peak (Apenten et al, 2002;Bonincontro et al, 2004;Brandts, 1964;Busti et al, 2005Busti et al, , 2006Chervenka, 1957;Donovan, 1969;Donovan, Laskowski, & Scheraga, 1958;Glazer & Smith, 1960;Laskowski, Widom, McFadden, & Scheraga, 1956;Poklar et al, 1993Poklar et al, , 1994Schmid, 2001).…”

Section: Quantitating the Denaturation Of Proteinsapplication Of Difference-uv Spectroscopymentioning

confidence: 99%

Effect of whey protein denaturation on the physicochemical properties of whey-based products

Nikolaidis¹,

Nikolaides²

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Μια καινοτόμος προσέγγιση στην ανάλυση φασμάτων διαφοράς υπεριώδους ακτινοβολίας αναπτύχθηκε για τον προσδιορισμό του βαθμού της θερμικής μετουσίωσης των πρωτεϊνών του ορού του γάλακτος. Η αλβουμίνη ορού ζωικής προέλευσης ήταν η πρώτη πρωτεΐνη που μελετήθηκε. Η ίδια μέθοδος, στη συνέχεια, εφαρμόστηκε στη μελέτη της επίδρασης της θέρμανσης, του pH, των υπερήχων και της αιθανόλης στη μετουσίωση μίγματος πρωτεϊνών του ορού του γάλακτος. Θεωρώντας τη δράση της υδροχλωρικής γουανιδίνης ως εκείνη που επιφέρει το μέγιστο ξεδίπλωμα (μετουσίωση) των πρωτεϊνών αυτών, βρέθηκε ότι ο μέγιστος βαθμός θερμικής μετουσίωσης της αλβουμίνης ήταν της τάξης του 17%, ενώ εκείνος του μίγματος των πρωτεϊνών του ορού γάλακτος, περίπου 40%. Η νέα αυτή μέθοδος ανάλυσης φασμάτων διαφοράς αποδείχθηκε ότι είναι ιδιαίτερα ευαίσθητη ακόμα και για συνδυασμούς θερμοκρασίας-χρόνου που διαφέρουν ελάχιστα μεταξύ τους. Η εφαρμογή της μεθόδου δεν απαιτεί διορθώσεις για τη σκέδαση του φωτός, ενώ επέτρεψε την πλήρη μελέτη της κινητικής της μετουσίωσης. Με βάση το εύρημα ότι η αιθανόλη προκαλεί 80% μετουσίωση του μίγματος των πρωτεϊνών του ορού γάλακτος, διπλάσιο ποσοστό συγκριτικά με εκείνο της θέρμανσης, σε ένα τελικό στάδιο της παρούσας μελέτης, μελετήθηκε η αντιστρεψιμότητα ή μη της μετουσίωσης που επιφέρει η αιθανόλη, εφαρμόζοντας διάφορες τεχνικές για την απομάκρυνση της αλκοόλης, όπως αραίωση με νερό, ξήρανση σε κλίβανο ή λυοφιλίωση. Στόχος ήταν να μελετηθεί η επίδραση της αιθανόλης στις λειτουργικές ιδιότητες των πρωτεϊνών ορού γάλακτος συγκριτικά με τη θέρμανση για πιθανή παραγωγή καινοτόμων προϊόντων. Με τη χρήση διαφορικής θερμιδομετρίας, συνεστιακής μικροσκοπίας και ρεολογικών μετρήσεων, βρέθηκε ότι οι πρωτεΐνες του ορού γάλακτος, μετά τη δράση της αιθανόλης και την απομάκρυνση αυτής στη συνέχεια, διατηρούν σε σημαντικό βαθμό το μετουσιωμένο χαρακτήρα τους (περίπου 34% διατήρηση) και ότι μπορούν να σχηματιστούν πηκτές απουσία θέρμανσης.

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Ultraviolet spectral changes during chymotrypsinogen activation

Cited by 19 publications

References 11 publications

Exposure of tyrosine residues in proteins. III. Reaction of cyanuric fluoride and N-acetylimidazole with ovalbumin, chymotrypsinogen, and trypsinogen

Exposure of tyrosine residues in proteins. III. Reaction of cyanuric fluoride and N-acetylimidazole with ovalbumin, chymotrypsinogen, and trypsinogen

The reactions of inter- and intra-chain disulphide bonds in proteins with sulphite

Effect of whey protein denaturation on the physicochemical properties of whey-based products

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